Variants of a Family 44 Xyloglucanase

ABSTRACT

The present invention relates to variants of a parent xyloglucanase. The present invention also relates to polynucleotides encoding the variant xyloglucanases and to nucleic acid constructs, vectors, and host cells comprising the polynucleotide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/193,478filed on Feb. 28, 2014 (pending, now allowed) which is a divisional ofU.S. application Ser. No. 12/995,706 filed on Dec. 14, 2010, now U.S.Pat. No. 8,709,777, which is a 35 U.S.C. 371 national application ofPCT/EP2009/056875 filed Jun. 4, 2009, which claims priority or thebenefit under 35 U.S.C. 119 of European application no. 08157769.4 filedJun. 6, 2008 and U.S. provisional application No. 61/059,832 filed Jun.9, 2008, the contents of which are fully incorporated herein byreference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to variants of a xyloglucanase belongingto family 44 of glycosyl hydrolases, polynucleotides encoding thevariants and methods of producing the variants.

BACKGROUND OF THE INVENTION

Xyloglucan is a major structural polysaccharide in the primary (growing)cell wall of plants. Structurally, xyloglucans consists of acellulose-like beta-1,4-linked glucose backbone which is frequentlysubstituted with various side chains. Xyloglucan is believed to functionin the primary wall of plants by cross-linking cellulose micro fibrils,forming a cellulose-xyloglucan network.

Xyloglucanses are capable of catalyzing the solubilization of xyloglucanto xyloglucan oligosaccharides. Some xyloglucanases only exhibitxyloglucanase activity, whereas others exhibit both xyloglucanase andcellulase activity. Xyloglucanses may be classified in EC 3.2.1.4 or EC.3.2.1.151. Enzymes with xyloglucanase activity are for example describedin Vincken et al., 1997, Carbohydrate Research 298(4): 299-310, whereinthree different endoglucanases EndoI, EndoV and EndoVI from Trichodermaviride (similar to T. reesei) are characterized. EndoI, EndoV and EndoVIbelongs to family 5, 7 and 12 of glycosyl hydrolases, respectively, seeHenrissat, 1991, Biochem. J. 280: 309-316, and Henrissat and Bairoch,1993, Biochem. J. 293: 781-788. WO 94/14953 discloses a family 12xyloglucanase (EG II) cloned from the fungus Aspergillus aculeatus. WO99/02663 discloses family 12 and family 5 xyloglucanases cloned fromBacillus licheniformis and Bacillus agaradhaerens, respectively. WO01/062903 discloses family 44 xyloglucanases.

In particular WO 99/02663 and WO 01/062903 suggest that xyloglucanasesmay be used in detergents.

It is an object of the present invention to provide variants ofxyloglucanases belonging to family 44 of glycosyl hydrolases withimproved properties compared to its parent enzyme.

SUMMARY OF THE INVENTION

The present invention relates to isolated variants of a parentxyloglucanase, comprising an alteration at one or more (several)positions selected from the group consisting of position number 68, 123,156, 118, 200, 129, 137, 193, 92, 83, 149, 34, 340, 332, 9, 76, 331,310, 324, 498, 395, 366, 1, 374, 7, 140, 8, 14, 21, 211, 37, 45, 13, 78,87, 436, 101, 104, 111, 306, 117, 119, 414, 139, 268, 142, 159, 164,102, 168, 176, 180, 482, 183, 202, 206, 217, 4, 222, 19, 224, 228, 232,2, 240, 244, 5, 247, 249, 328, 252, 259, 406, 267, 269, 275, 179, 166,278, 281, 288, 298, 301, 18, 302, 165, 80, 303, 316, 169, 322, 120, 146,342, 348, 147, 353, 380, 468, 382, 383, 38, 384, 389, 391, 10, 392, 396,177, 397, 399, 409, 237, 413, 253, 415, 418, 40, 443, 445, 148, 449,225, 450, 454, 3, 455, 456, 299, 461, 470, 204, 476, 488, 347, and 507,which position corresponds to a position in amino acid sequence SEQ IDNO:3

wherein the alteration(s) are independently

i) an insertion of an amino acid downstream of the amino acid whichoccupies the position,

ii) deletion of the amino acid which occupies the position, or

iii) a substitution of the amino acid which occupies the position with adifferent amino acid; and the parent xyloglucanase is a family 44xyloglucanase; and the variant has xyloglucanase activity.

The present invention also relates to isolated polynucleotides encodingthe variant xyloglucanases or polypeptides having xyloglucanasesactivity, nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides, and methods of producing a variant of a parentxyloglucanase or a polypeptide having xyloglucanases activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1H shows consensus sequences performed by aligning SEQ IDNO: 3, with SEQ ID NO: 5 and SEQ ID NO: 7 as well as with othersequences from the uniprot database which are 30% identical to thefamily 44 glycosyl hydrolase region of SEQ ID NO: 3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to variants of parent family 44xyloglucanases, comprising an alteration, preferably in the form of asubstitution and/or an insertion and/or a deletion at one or more(several) positions, where the numbering of the positions corresponds tothe numbering of the positions of SEQ ID NO:3. The variants of thepresent invention have xyloglucanase activity and potentially alsocellulolytic activity. The variants of the present invention haveimproved properties compared to the parental xyloglucanase. In oneaspect, the variants have improved stability in liquid detergents,especially liquid laundry detergent compositions.

DEFINITIONS

Xyloglucanase activity: The term “xyloglucanase activity” is definedherein as an enzyme catalyzed hydrolysis of xyloglucan. The reactioninvolves endo hydrolysis of 1,4-beta-D-glucosidic linkages inxyloglucan. For purposes of the present invention, xyloglucanaseactivity is determined using AZCL-xyloglucan (from Megazyme) as thereaction substrate. The assay can be performed in several ways, e.g. asdescribed in Example 2 of the present application or as described in WO01/62903. One unit of xyloglucanase activity (XyloU) is defined byreference to the assay method described in WO 01/62903, page 60, lines3-17.

Cellulase activity: The term “cellulase activity” is defined herein asan enzyme catalyzed hydrolysis of 1,4-beta-D-glucosidic linkages inbeta-1,4-glucan (cellulose). For purposes of the present invention,cellulase activity is determined using AZCL-HE-cellulose (from Megazyme)as the reaction substrate.

Variant: The term “variant” is defined herein as a polypeptide havingxyloglucanase activity comprising an alteration, such as a substitution,insertion, and/or deletion, of one or more (several) amino acid residuesat one or more (several) specific positions which positions correspondto the amino acid positions in SEQ ID NO: 3. The variants of theinvention may also have cellulase activity. The altered polypeptide(variant) is obtained through human intervention by modification of thepolynucleotide sequence encoding the parental enzyme. The parentalenzyme may be encoded by SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 6 or asequence which is at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95% identical to one of these sequences and which encode an activepolypeptide. The variant polypeptide sequence is preferably one which isnot found in nature.

Wild-Type Enzyme: The term “wild-type” xyloglucanase denotes axyloglucanase expressed by a naturally occurring microorganism, such asbacteria, yeast, or filamentous fungus found in nature. The termwild-type may be used interchangeably with the term “naturallyoccurring”.

Parent Enzyme: The term “parent” xyloglucanase or “parental”xyloglucanase as used herein means a xyloglucanase to which amodification, e.g., substitution(s), insertion(s), deletion(s), and/ortruncation(s), is made to produce the enzyme variants of the presentinvention. This term also refers to the polypeptide with which a variantis compared and aligned. The parent may be a naturally occurring(wild-type) polypeptide such as the enzyme of SEQ ID NO:2 or SEQ ID NO:3or SEQ ID NO: 5 or SEQ ID NO: 7 or a polypeptide which is at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95% identical to oneof these sequences. The parent polypeptide may also be a variant of anaturally occurring polypeptide which has been modified or altered inthe amino acid sequence. A parent may also be an allelic variant, whichis a polypeptide encoded by any of two or more alternative forms of agene occupying the same chromosomal locus.

Isolated variant or polypeptide: The term “isolated variant” or“isolated polypeptide” as used herein refers to a variant or apolypeptide that is isolated from a source, e.g. the host cell fromwhich it is expressed or the enzyme complex it is normally present in.Preferably, the polypeptide is at least 40% pure, more preferably atleast 60% pure, even more preferably at least 80% pure, most preferablyat least 90% pure, and even most preferably at least 95% pure, asdetermined by SDS-PAGE.

Substantially pure variant or polypeptide: The term “substantially purevariant” or “substantially pure polypeptide” denotes herein apolypeptide preparation that contains at most 10%, preferably at most8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polypeptide material with which it is nativelyor recombinantly associated. It is, therefore, preferred that thesubstantially pure variant or polypeptide is at least 92% pure,preferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 96% pure, morepreferably at least 97% pure, more preferably at least 98% pure, evenmore preferably at least 99%, most preferably at least 99.5% pure, andeven most preferably 100% pure by weight of the total polypeptidematerial present in the preparation. The variants and polypeptides ofthe present invention are preferably in a substantially pure form. Thiscan be accomplished, for example, by preparing the variant orpolypeptide by well-known recombinant methods or by classicalpurification methods.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having xyloglucanase activity that is in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc. For the polypeptide defined by SEQ ID NO: 2, themature xyloglucanase sequence may in theory start at position 28 of SEQID NO: 2. The mature sequence ends at position 551 of SEQ ID NO: 2. Thetheoretical mature xyloglucanase sequence is show in SEQ ID NO: 3.Depending on expression system the length of the actual maturepolypeptide may vary 1 to 10 amino acids in length based on thetheoretical mature polypeptide. The mature polypeptide may for examplestart at position 33 of SEQ ID NO: 2 and ends at position 551 of SEQ IDNO: 2.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having xyloglucanase activity. In one aspect, themature polypeptide coding sequence is nucleotides 82 to 1653 of SEQ IDNO: 1. The mature polypeptide coding sequence may vary 3 to 30nucleotides in length depending on the expression system. The maturepolypeptide coding sequence can for example correspond to nucleotides 97to 1653 of SEQ ID NO: 1.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends in Genetics 16: 276-277; emboss.org), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra; emboss.org), preferably version 3.0.0 or later. The optionalparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitutionmatrix. The output of Needle labeled “longest identity” (obtained usingthe—nobrief option) is used as the percent identity and is calculated asfollows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Functional fragment: The term “functional fragment of a polypeptide” isused to describe a polypeptide which is derived from a longerpolypeptide, e.g., a mature polypeptide, and which has been truncatedeither in the N-terminal region or the C-terminal region or in bothregions to generate a fragment of the parent polypeptide. To be afunctional polypeptide the fragment must maintain at least 20%,preferably at least 40%, more preferably at least 50%, more preferablyat least 60%, more preferably at least 70%, more preferably at least80%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 100% of the xyloglucanase activity ofthe full-length/mature polypeptide.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide that is isolated from a source. In oneaspect, the isolated polynucleotide is at least 40% pure, morepreferably at least 60% pure, even more preferably at least 80% pure,and most preferably at least 90% pure, and even most preferably at least95% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered polypeptide production systems.Thus, a substantially pure polynucleotide contains at most 10%,preferably at most 8%, more preferably at most 6%, more preferably atmost 5%, more preferably at most 4%, more preferably at most 3%, evenmore preferably at most 2%, most preferably at most 1%, and even mostpreferably at most 0.5% by weight of other polynucleotide material withwhich it is natively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99%, and even most preferably at least 99.5%pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively or recombinantly associated. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

Coding sequence: When used herein the term “coding sequence” means apolynucleotide, which directly specifies the amino acid sequence of itspolypeptide product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant polynucleotide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Host cell: The term “host cell”, as used herein, includes any cell typethat is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or a vector comprising apolynucleotide of the present invention. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication.

Improved chemical stability: The term “improved chemical stability” isdefined herein as a variant enzyme displaying retention of enzymaticactivity after a period of incubation in the presence of a chemical orchemicals, either naturally occurring or synthetic, which reduces theenzymatic activity of the parent enzyme. Improved chemical stability mayalso result in variants better able to catalyze a reaction in thepresence of such chemicals. In a particular aspect of the invention theimproved chemical stability is an improved stability in a detergent, inparticular in a liquid detergent. The improved detergent stability is inparticular an improved stability of the xyloglucanase activity when axyloglucanase variant of the present invention is mixed into a liquiddetergent formulation and then stored at temperatures between 15 and 50°C.

In the present invention liquid detergents are particular useful asliquid laundry detergents.

Conventions for Designation of Variants

For purposes of the present invention, the amino acid sequence of thexyloglucanase disclosed in SEQ ID NO: 3 is used to determine thecorresponding amino acid residue in another xyloglucanase. The aminoacid sequence of another xyloglucanase is aligned with the amino acidsequence of the xyloglucanase disclosed in SEQ ID NO: 3, and based onthe alignment the amino acid position number corresponding to any aminoacid residue in the amino acid sequence of the xyloglucanase disclosedin SEQ ID NO: 3 can be determined.

An alignment of polypeptide sequences may be made, for example, using“ClustalW” (Thompson et al., 1994, CLUSTAL W: Improving the sensitivityof progressive multiple sequence alignment through sequence weighting,positions-specific gap penalties and weight matrix choice, Nucleic AcidsResearch 22: 4673-4680). An alignment of DNA sequences may be done usingthe polypeptide alignment as a template, replacing the amino acids withthe corresponding codon from the DNA sequence.

In describing the various xyloglucanase variants of the presentinvention, the nomenclature described below is adapted for ease ofreference. In all cases, the accepted IUPAC single letter or tripleletter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: original amino acid/position/substituted aminoacid. Accordingly, the substitution of threonine with alanine atposition 226 is designated as “Thr226Ala” or “T226A”. Multiple mutationsare separated by addition marks (“+”), e.g., “G205R+S411F”, representingmutations at positions 205 and 411 substituting glycine (G) witharginine (R), and serine (S) with phenylalanine (F), respectively. Wherean original amino acid may be substituted by an amino acid selected froma group it is designated as “K129R,S,A,I,F,Q” representing thesubstitution of a lysine (K) at position 129 with an amino acid selectedfrom the group consisting of: arginine (R), serine (S), alanine (A),isoleucine (I), phenylalanine (F) and glutamine (Q). Alternatively,“K129R,S,A,I,F,Q” could be written as K129R or K129S, or K129A, or K129Ior K129F or K129Q

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid/position/asterisk (*). Accordingly, thedeletion of glycine at position 195 is designated as “Gly195*” or“G195*”. Multiple deletions are separated by addition marks (“+”), e.g.G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Asterisk (*)/position/lower case letter/inserted amino acid, wherethe lower case letter indicates the addition of an amino acid downstream of the position number. Accordingly, the insertion of a glutamicacid (E) down stream of position 10 is designated “*10aE”. If a secondamino acid, e.g. a valine (V), is to be inserted down stream of position10 after the glutamic acid (E) it is designated “*10aE+*10bV”. Additionsto the N-terminal of the polypeptide are designated with a 0 (zero). Theaddition of a glutamic acid (E) and a valine (V) added to the N-terminalamino acid of a polypeptide is designated as *0aE+*0bV. A “downstream”insertion can also be described as the addition of one or more aminoacids between the named position and the position immediately followingthe named position, e.g. an insertion downstream of position 195 resultsin the addition of one or more amino acids between position 195 and 196,thereby generating new positions *195a, *195b and so forth.

Parent Xyloglucanases

In the present invention, the parent xyloglucanase is either (a) axyloglucanase belonging to family 44 of glycosyl hydrolases also termedfamily 44 xyloglucanases; or (b) a polypeptide selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO: 5 and SEQ ID NO: 7; or (c) apolypeptide comprising an amino acid sequence having at least 75%identity with the mature polypeptide of SEQ ID NO: 3; or (d) apolypeptide encoded by a polynucleotide that hybridizes under at leastmedium stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 6, (ii) thegenomic DNA sequence comprising the mature polypeptide coding sequenceof SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 6 or (iii) a full-lengthcomplementary strand of (i) or (ii); or (e) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 70%identity with the mature polypeptide coding sequence of SEQ ID NO: 1.

In a first aspect, the parent xyloglucanase comprise an amino acidsequence having a degree of identity to the mature polypeptide of SEQ IDNO: 3 of preferably at least at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 95%, more preferablyat least 96%, even more preferably at least 97%, most preferably atleast 98%, or even most preferably at least 99%, which havexyloglucanase activity (hereinafter “homologous polypeptides”). In oneaspect, the homologous polypeptides have an amino acid sequence thatdiffers by ten amino acids, preferably by nine, more preferably byeight, more preferably by seven, more preferably by six, more preferablyby five amino acids, more preferably by four amino acids, even morepreferably by three amino acids, most preferably by two amino acids, andeven most preferably by one amino acid from the mature polypeptide ofSEQ ID NO: 3.

Substantially homologous parent xyloglucanases may have one or more(several) amino acid alterations such as substitutions, deletions and/orinsertions. These changes are preferably of a minor nature, that isconservative amino acid substitutions and other substitutions that donot significantly affect the three-dimensional folding or activity ofthe protein or polypeptide; small deletions, typically of one to about 9amino acids, preferably from one to about 15 amino acids and mostpreferably from one to about 30 amino acids; and small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, a small linker peptide of up to about five to ten residues,preferably from 10 to 15 residues and most preferably from 20 to 25residues, or a small extension that facilitates purification (anaffinity tag), such as a poly-histidine tag, or protein A (Nilsson etal., 1985, EMBO J. 4: 1075; Nilsson et al., 1991, Methods Enzymol. 198:3). See, also, in general, Ford et al., 1991, Protein Expression andPurification 2: 95-107.

Although the changes described above preferably are of a minor nature,such changes may also be of a substantive nature such as fusion oflarger polypeptides of up to 300 amino acids or more both as amino- orcarboxyl-terminal extensions.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by Neurath and Hill,1979, In, The Proteins, Academic Press, New York. The most commonlyoccurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Essential amino acids in the xyloglucanase polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-1085, 1989). In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (i.e. xyloglucanase activity) to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306-312,1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al.,FEBS Lett. 309:59-64, 1992. The identities of essential amino acids canalso be inferred from analysis of homologies with polypeptides which arerelated to a polypeptide according to the invention. The crystalstructure of an enzyme belonging to the family 44 glycosyl hydrolaseshas been published by Kitago et al., 2007, J. Biol. Chem. 282:35703-35711. Based on this structure it is possible to generate a threedimensional structure of the parent xyloglucanase (SEQ ID NO: 3) insilico. Based on comparison with the published structure the followingresidues in SEQ ID NO: 3 have been identified as critical for theenzymatic function E187 (Catalytic-Acid/Base), E358(Catalytic—Nucleophile), E56 (Carboxylate group coordinating Ca2+) andD154 (Carboxylate group coordinating Ca2+). These positions should,therefore, preferably not be mutated in the parent enzyme.

The parent xyloglucanase preferably comprises the amino acid sequence ofSEQ ID NO: 3 or an allelic variant thereof; or a fragment thereof havingxyloglucanases activity. In one aspect, the parent xyloglucanasecomprises the amino acid sequence of SEQ ID NO: 2. In another aspect,the parent xyloglucanase comprises the mature polypeptide of SEQ ID NO:2. In another aspect, the parent xyloglucanase consists of the aminoacid sequence of SEQ ID NO: 3 or an allelic variant thereof; or afragment thereof having xyloglucanase activity. In another aspect, theparent xyloglucanase comprises the amino acid sequence of SEQ ID NO: 5,or an allelic variant thereof; or a fragment thereof havingxyloglucanase activity. In another aspect, the parent xyloglucanasecomprises the amino acid sequence of SEQ ID NO: 7, or an allelic variantthereof; or a fragment thereof having xyloglucanase activity. In anotheraspect the parent xyloglucanase comprises an amino acid sequence whichis at least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, evenmore preferably at least 90%, most preferably at least 95% identical SEQID NO: 2, or SEQ ID NO: 3 or SEQ ID NO: 5.A fragment of the maturepolypeptide of SEQ ID NO: 3 is a polypeptide having one or more(several) amino acids deleted from the amino- and/or carboxyl-terminusof this amino acid sequence and still maintaining xyloglucanaseactivity.

In a second aspect, the parent xyloglucanases are encoded bypolynucleotides that hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 6, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:1 or SEQ ID NO: 4 or SEQ ID NO: 6, (iii) a subsequence of (i) or (ii),or (iv) a full-length complementary strand of (i), (ii), or (iii) (J.Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Thesubsequence may encode a polypeptide fragment having xyloglucanaseactivity. In one aspect, the complementary strand is the full-lengthcomplementary strand of the mature polypeptide coding sequence of SEQ IDNO: 1 or SEQ ID NO: 4 or SEQ ID NO: 6.

A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 1or SEQ ID NO: 4 or SEQ ID NO: 6, or a homolog thereof, is a nucleotidesequence where one or more (several) nucleotides have been deleted fromthe 5′- and/or 3′-end, where the polypeptide encoded by the subsequencepossess xyloglucanase activity.

The parent enzymes may also be allelic variants of the polypeptides thathave xyloglucanase activity.

The polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 6; or asubsequence thereof; as well as the amino acid sequence of SEQ ID NO: 3or SEQ ID NO: 5 or SEQ ID NO: 7; or a fragment thereof; may be used todesign nucleic acid probes to identify and clone DNA encoding parentxyloglucanases from strains of different genera or species according tomethods well known in the art. In particular, such probes can be usedfor hybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least14, preferably at least 25, more preferably at least 35, and mostpreferably at least 70 nucleotides in length. It is, however, preferredthat the nucleic acid probe is at least 100 nucleotides in length. Forexample, the nucleic acid probe may be at least 200 nucleotides,preferably at least 300 nucleotides, more preferably at least 400nucleotides, or most preferably at least 500 nucleotides in length. Evenlonger probes may be used, e.g., nucleic acid probes that are preferablyat least 600 nucleotides, more preferably at least 700 nucleotides, evenmore preferably at least 800 nucleotides, preferably at least 900nucleotides in length, preferably at least 1000 nucleotides in length,preferably at least 1100 nucleotides in length, preferably at least 1200nucleotides in length, preferably at least 1300 nucleotides in length,preferably at least 1400 nucleotides in length, preferably at least 1500nucleotides in length or most preferably at least 1600 nucleotides inlength. Both DNA and RNA probes can be used. The probes are typicallylabeled for detecting the corresponding gene (for example, with ³²P, ³H,³⁵5, biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA library prepared from other organisms may be screened forDNA that hybridizes with the probes described above and encodes a parentxyloglucanase. Genomic or other DNA from other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that is homologouswith SEQ ID NO: 1, or a subsequence thereof, the carrier material isused in a Southern blot. For purposes of the present invention,hybridization indicates that the polynucleotide hybridizes to a labelednucleotide probe corresponding to the polynucleotide shown in SEQ ID NO:1, its complementary strand, or a subsequence thereof, under low to veryhigh stringency conditions. Molecules to which the probe hybridizes canbe detected using, for example, X-ray film or any other detection meansknown in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe isnucleotides 82 to 1653 of SEQ ID NO: 1, or nucleotides 97 to 1653 of SEQID NO: 1. In another aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 2, or a subsequencethereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at 45° C. (very low stringency), more preferably at50° C. (low stringency), more preferably at 55° C. (medium stringency),more preferably at 60° C. (medium-high stringency), even more preferablyat 65° C. (high stringency), and most preferably at 70° C. (very highstringency).

For short probes that are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes that are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third aspect, the parent xyloglucanase is encoded by apolynucleotide comprising or consisting of a nucleotide sequence havinga degree of identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably 96%, 97%, 98%, or 99%, which encodean active polypeptide. In one aspect, the mature polypeptide codingsequence is nucleotides 82 to 1653 of SEQ ID NO: 1, or nucleotides 97 to1653 of SEQ ID NO: 1.

The parent xyloglucanase may be obtained from microorganisms of anygenus. In one aspect, the parent xyloglucanase is secretedextracellularly.

In a further aspect the parent xyloglucanase may be a bacterialxyloglucanase. For example, the xyloglucanase may be a Gram positivebacterial polypeptide such as a Bacillus, preferably from theBacillus/Lactobacillus subdivision, preferably a species from the genusPaenibacillus, especially Paenibacillus polymyxa, e.g. Paenibacilluspolymyxa, ATCC 832, preferably the xyloglucanase is a family 44xyloglucanse, e.g. as described in WO 01/62903, more preferably thexyloglucanase of SEQ ID NO: 5, more preferably the xyloglucanase of SEQID NO: 7, and most preferably the xyloglucanase of SEQ ID NO: 2 or themature polypeptide thereof.

Generation of Variants

Variants of a parent xyloglucanase can be prepared according to anymutagenesis procedure known in the art, such as random and/orsite-directed mutagenesis, synthetic gene construction, semi-syntheticgene construction, random mutagenesis, shuffling, etc.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide molecule of interest.Gene synthesis can be performed utilizing a number of techniques, suchas the multiplex microchip-based technology described by Tian et al.,(Tian, et. al., Nature 432:1050-1054) and similar technologies whereinoligonucleotides are synthesized and assembled upon photo-programmablemicrofluidic chips.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide fragments may then be shuffled.

Site-directed mutagenesis is a technique in which one or severalmutations are created at a defined site in a polynucleotide moleculeencoding the parent xyloglucanase. The technique can be performed invitro or in vivo.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parentxyloglucanase and subsequent ligation of an oligonucleotide containingthe mutation in the polynucleotide. Usually the restriction enzyme thatdigests at the plasmid and the oligonucleotide is the same, permittingsticky ends of the plasmid and insert to ligate to one another. Forfurther description of suitable techniques reference is made to Sambrooket al. (1989), Molecular cloning: A laboratory manual, Cold SpringHarbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.)“Current protocols in Molecular Biology”. John Wiley and Sons, 1995;Harwood, C. R., and Cutting, S. M. (eds.) “Molecular Biological Methodsfor Bacillus”. John Wiley and Sons, 1990), and WO 96/34946; Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Research 18: 7349-4966.

After the ligase reaction the ligation mixture may be used to transforma host cell, for cloning purposes E. coli cells are often used asdescribed in Ausubel, F. M. et al. The transformed E. coli cells can bepropagated in liquid media or on solid agar plates, plasmids can berescued from the transformed cells and used to transform B. subtiliscells. Suitable competent Bacillus cells, such as MB1510, a168-derivative (e.g. available from BGSC with accession no. 1A1 168trpC2), may be transformed as described in WO 03/095658. An E. coliplasmid-borne integration cassette for library construction may be usedfor Bacillus transformation. The method is described in detail in WO03/095658. Alternatively, an in vitro amplified PCR-SOE-product(Melnikov and Youngman, Nucleic Acid Research 27, 1056) may be used.

Site-directed mutagenesis can be accomplished in vivo by methods knownin the art. See, for example, U.S. Patent Application Publication2004/0171154; Storici et al., 2001, Nature Biotechnology 19: 773-776;Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants of a parent xyloglucanases.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochem. 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Neret al., 1988, DNA 7:127).

Mutagenesis/shuffling methods as described above can be combined withhigh-throughput, automated screening methods to detect the activity ofcloned, mutagenized polypeptides expressed by host cells, e.g. Bacillusas described above. Mutagenized DNA molecules that encode polypeptidesweith xyloglucanase activity can be recovered from the host cells andrapidly sequenced using standard methods in the art.

Variants

In the present invention, the isolated variants of a parentxyloglucanase comprise an alteration at one or more (several) positionsselected from the group consisting of positions number 68, 123, 156,118, 200, 129, 137, 193, 92, 83, 149, 34, 340, 332, 9, 76, 331, 310,324, 498, 395, 366, 1, 374, 7, 140, 8, 14, 21, 211, 37, 45, 13, 78, 87,436, 101, 104, 111, 306, 117, 119, 414, 139, 268, 142, 159, 164, 102,168, 176, 180, 482, 183, 202, 206, 217, 4, 222, 19, 224, 228, 232, 2,240, 244, 5, 247, 249, 328, 252, 259, 406, 267, 269, 275, 179, 166, 278,281, 288, 298, 301, 18, 302, 165, 80, 303, 316, 169, 322, 120, 146, 342,348, 147, 353, 380, 468, 382, 383, 38, 384, 389, 391, 10, 392, 396, 177,397, 399, 409, 237, 413, 253, 415, 418, 40, 443, 445, 148, 449, 225,450, 454, 3, 455, 456, 299, 461, 470, 204, 476, 488, 347, and 507,wherein the variant having xyloglucanase activity comprises an aminoacid sequence having a degree of identity of at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, more preferably atleast 95%, more preferably at least about 97%, most preferably at least98% and even more preferably 99% to the amino acid sequence of theparent xyloglucanase. The numbering of the positions are relative to theamino acid sequence of SEQ ID NO: 3. Preferably, the variants comprisingalterations at one or more of the above identified positions have anincreased stability in detergent, preferably in liquid detergent ascompared to the parent xyloglucanase.

In a preferred embodiment the variant comprises one or more (several) ofthe following combinations of alterations:

V1*+V2*+H3*; V1Q+*1aE+*1bV; H3A; H3A+H436A; K8A,Q,S; T9D;T9D+L34F+A83E+Q149E+H193T+S332P+R340T;

I10V+D33E+M40L+A41T+Q67M+Y73F+S76D+G78A+Q82K+T92A+L102Q+Q137E+I222V+V228I+D249N+S269N+V272A+E333A+I337L+M356L+T374A+S416A+D444Y+A469E+K470T+I473G+T517A+S522*;I10V+F17S+D33E+M40L+A41T+Q67M+N72S+S76D+G78A+Q82K+Q137E+V219A+D249N+V272A+1337L+M356L+V397A+S416A+T421I+S424N+N441D+D444Y+V450I+K470T+I473S+V477I;I10V+F17S+D33E+M40L+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+Q137E+H164N+N168K+T172A+V219A+I222V+V228I+D249N+S269N+V272A+E333A+I337L+M356L+N415S+T421I+S424H+N441D+D444Y+S522P+P523V+V524E;I10V+F17S+D33E+M40L+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+Q137E+I222V+V228I+D249N+V272A+I337L+M356L+T374A+V397A+S416A+T421I+S424N+N441D+D444Y+V450I+A469E+K470T+I473G+T517A+S522P+P523V+V524E;I10V+F17S+D33E+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+Q137E+N168K+T172A+I222V+V228I+D249N+V272A+E333A+I337L+M356L+V397A+S416A+T421I+S424H+N441D+D444Y+A469E+K470T+I473S+V477I+E489A+A490V+T517A+S522*;I10V+F17S+M40L+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+Q137E+I222V+V228I+D249N+S269N+V272A+T320A+I337L+M356L+T374A+V397A+N415S+T421I+S424H+N441D+D444Y+A469E+K470T+I473S+V477I+T517A+S522P+P523V+V524E;I10V+F17S+Q67M+N72S+S76D+G78A+Q82K+T104A+Q137E+N153K+R156Q+V219A+I222V+V228I+D249N+S269N+V272A+E333A+I337L+M356L+V397A+N415S+D420G+T421I+S424H+N441D+D444Y+V450I+A469E+K470T+I473G+T517A+S522*;

I10V+F17S+Q67M+N72S+S76D+G78A+Q82K+T92A+T104A+Q137E+R156Q+V159A+H164N+N168K+T172A+I222V+V228I+D249N+V272A;

I10V+F17S+Y53H+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+Q137E+T172V+A177T+I222V+V228I+D249N+S269N+I337L+M356L+V397A+S416A+T421I+S424H+N441D+D444Y+A469E+K470T+I473G+T517A+S522*;

K13A+K129A; K13A+Q68H+T92V+K118A+Q137E+R156Y+G200P; K13A,R; K18R; R20A;K21Q+K129A; K21Q,R,T;Q32H+M40L+R49G+D65E+Q67M+N72S+S76D+G78A+Q82K+T92A+L102Q+T104A+Q137E+H164N+K202E+I222V+V228I+D249N+M356L+T374A;D33V+Q68H+FN168H+V450I; L34F,I,M,V; L34I+K129A; D37G,N+K129A+R156Y;E38I,V;M40L+A41T+Q67M+N72S+S76D+G78A+Q82K+Q137E+N153K+H164N+D249N+V272A+I337L+M356L+V397A+N415S+T421I+S424N+N441D+V450I+E489A+A490V+T517A+S522*;M40V; L451; Q68H,M,N; Q68H+G200P+N331F;Q68H+K118A+K129A+R156Y+G200P+N331F; Q68H+K118A+R156V+G200P+N331F;Q68H+K118A+R156Y+H193T+D366H; Q68H+K118R+R156F,Y;Q68H+K118R+R156Y+G200P; Q68H+K118S+R156F+G200P+G274D+N331F;Q68H+K129A,T+R156K+G200P+N331F; Q68H+R156F,V,Y+G200P+N331F; Q68H+R156Y;Q68H+R156Y+H193T; Q68H+R156Y+H193T+D366H; Q68H+R156Y+H193T+G200P+M310V;Q68H+S76W+T92V+K118A+Q137E+R156Y+G200P+N331F;Q68H+T92A,D,I,S,V,Y+K118A+K129A+R156Y+G200P+N331F;Q68H+T92N+D97N+K118A+K129A+R156Y+G200P+N331F;Q68H+T92S+K118A+K129A+R156Y+G200P+G274D+N331F; Q68H+T92V+G200P+M310V;Q68H+T92V+G200P+M310V+N331F;Q68H+T92V+K118A+K129A+Q137E+R156Y+G200P+A224P+N331F;Q68H+T92V+K118A+K129A+Q137E+R156Y+G200P+N331F;Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T;Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+D366H;Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+G200P+M310V+E446K;Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+N331H,K,Q;Q68H+T92V+K118A+K129A+R156Y+H193T;Q68H+T92V+K118A+K129A+R156Y+H193T+D366H;Q68H+T92V+K118A+K129A+R156Y+H193T+G200P+M310V;Q68H+T92V+K118A+Q137E+N140F+R156Y+G200P+K470T;Q68H+T92V+K118A+Q137E+R156Y+G200P+D324N;Q68H+T92V+K118A+Q137E+R156Y+G200P+K470T;Q68H+T92V+K118A+Q137E+R156Y+G200P+M310L;Q68H+T92V+K118A+Q137E+R156Y+G200P+N331F; Q68H+T92V+K118A,R+R156Y,F;Q68H+T92V+K118A+S123P,T+K129A+Q137E+R156Y+G200P+N331F;Q68H+T92V+K118R+R156Y+H193T+D366H; Q68H+T92V+R156F+G200P+M310V+S484C;Q68H+T92V+R156F,V,Y+G200P+M310V; Q68H+T92V+R156F,V,Y+G200P+M310V+N331F;Q68H+T92V+R156F,Y+H193T; Q68H+T92V+R156F,Y+H193T+D366H;Q68H+T92V+R156F,Y+H193T+G200P+M310V; Q68H+T92V+R156Y;S76E,I,K,M,R,T,V,W; S76W+G200P; S76W+G200P+A224P;G78A+K118A++K129A+R156Y; G78A+K118A+K129A+R156Y;G78A+K118A+K129A+R156Y+G200P+N331F; G78A+K118A+K129A+R156Y+K169A;G78A,N,S; G78A+T92V+K118A+K129A+R156Y;G78A+T92V+K118A+K129A+R156Y+G200P+N331F;G78A+T92V+K118A+K129A+R156Y+K169A; L80V; A83D,E,H,I,L,N,R,S,T,Y; K87Q;K87V+K129A+K169A; T921,V; T92V+K118A+K129A+Q137E+R156Y+G200P+N331F;T92V+K118A+K129A+R156Y; T92V+K118A+K129A+R156Y+G200P+N331F;T92V+K118A+K129A+R156Y+H164N+G200P+N331F; T92V+K129A+R156Y; K101A+K129A;K101R; K101R+L1021; T104A+P111Q+A117S+K129A+R156Y; P111Q; K118A+K129A;K118A+K129A+F146L+R156Y+G200P+N331F;K118A+K129A+Q137E+R156Y+G200P+N331F; K118A+K129A+R156Y;K118A+K129A+R156Y+A224P; K118A+K129A+R156Y+G200P;K118A+K129A+R156Y+G200P+M310V+N331F; K118A+K129A+R156Y+G200P+N331F;K118A+K129A+R156Y+G200P+N331F+N3991;K118A+K129A+R156Y+K169A+G200P+N331F; K118A+K129A+R156Y+K470T; K118A,R;K118A+R156Y; K118A+R156Y+G200P; D119L; G120A; S123P,T;S123T+K129A+R156Y; K129A,F,I,K,R,S,T; K129A+K169A; K129A+K176P;K129A+K275Q; K129A+K445S; K129A+K470T; K129A+Q137E+R156Y;K129A+Q137E+R156Y+G200P; K129A+Q137E+R156Y+K470T;K129A+Q137E+V139K+N140F+Q147S+R156Y; K129A+R156Y;K129A+R156Y+A177T+V179I+A183S; K129A+R156Y+A328G; K129A+R156Y+D247G;K129A+R156Y+D249G,N,S; K129A+R156Y+D3031,K,S,V; K129A+R156Y+D324N;K129A+R156Y+D366H+T374A; K129A+R156Y+D461N,Q,T; K129A+R156Y+E288Q;K129A+R156Y+G200P; K129A+R156Y+G200P+G204T+R211K; K129A+R156Y+H164N;K129A+R156Y+H436Y; K129A+R156Y+I10V+V14I+D19E;K129A+R156Y+I222V+A224P+V228I+V232A; K129A+R156Y+K176P,S;K129A+R156Y+K275T; K129A+R156Y+K322I+K454Q; K129A+R156Y+K406N+N415G;K129A+R156Y+K454Q; K129A+R156Y+L380F+N383Y+D384G+N389T;K129A+R156Y+N298F+E299N+G301T; K129A+R156Y+N302K+D303L,S;K129A+R156Y+N331F; K129A+R156Y+P507A; K129A+R156Y+R267H;K129A+R156Y+R409L,T; K129A+R156Y+S443D+K445S+L449I+V450I+S455N+M456Y;K129A+R156Y+T244D; K129A+R156Y+V159M+H164N+F165Y;K129A+R156Y+V259I+R267K+L268K+S269A; Q137D,E; N140F; K142A,Q,R;F146C+H164C; F146K,L; F146L+K322I; L148K+N168D; Q149E;R156A,D,E,F,I,K,L,M,N,P,Q,R,S,T,V,W,Y; R56Y+N331F; V159M; H164A,N;L1661; N168D; K169A,Q,R; K176P; A177E,T; K180R; H193A,D,S,T; R197A,L;H199A; G200A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; G200P+A224P;K202N,Q,R; S214E; K217A; A221K; G225S; V232A; G237A,S,V; K240A,Q,R;K252A,Q,R; G253A; R267A; L2681; K275A,Q,R; L2781; F281 L; M290R; R295A;K306A,R; K307Q; M310I,L,V; M310V+N3991; R314A; G3161; K322A,R; D324N;N331A,C,D,E,F,G,H,I,K,L,M,P,Q,R,S,T,V,W,Y; S332M,P; S332P+V3971;R340A,N,T; K342A; V3451; K347A,Q,R; D348G; K353Q,R; D366H; M373Q; T374A;L380F; K382A; N383Y; N389A,F,N,V; W391V; K392G,Q; D395G; G396P; V397S;N3991; K406N; G413A,S; K414A; N415S; T417K; F4181; V431E; H436A;N441G+A442E+S443D; S443E,K,Q; K445A,R,S; K445C+K470C; H448A; K454R;S467R+G468S+A469T; G468S,Y; K470P,R,T; 1473T; K476Q; K482A,Q,R;K488A,Q,R,T; A490R; G498A,D,S; R500A,T,V; H512A; T517A+G518D; or G518D;

In one aspect, the number of amino acid alterations in the variants ofthe present invention comprise preferably the total number of 55,preferably 52, more preferably 50, more preferably 40, more preferably30, more preferably 20, more preferably 15, more preferably ten, morepreferably nine, more preferably eight, even more preferably seven, evenmore preferably six, even more preferably five, even more preferablyfour, even more preferably three, and most preferably two alterations,and most preferably one alteration. In another aspect the total numberof alterations is one, preferably two, more preferably three, even morepreferably four, even more preferably five, even more preferably six,even more preferably seven, even more preferably eight, even morepreferably nine, most preferably ten. The alteration may be in the formof i) an insertion of an amino acid downstream of the amino acid whichoccupies the position; ii) deletion of the amino acid which occupies theposition, or iii) a substitution of the amino acid which occupies theposition with a different amino acid. The alterations may be madeindependently of each other, for example in one position there may be aninsertion while there is a substitution at a second position and adeletion at a third position as compared to the parental xyloglucanase.In a preferred embodiment the variant only comprises substitutions.

In one aspect of the invention positions to be mutated are identifiedbased on consensus sequence analysis. The analysis is performed byaligning SEQ ID NO: 3, with SEQ ID NO: 5 and SEQ ID NO: 7 as well aswith other sequences from the uniprot database which are 30% identicalto the family 44 glycosyl hydrolase region of SEQ ID NO: 3. Theresulting consensus sequences are shown in FIG. 1. Consensus sequence 1is the sequence comprising the most abundant amino acid at a givenposition from the alignment, consensus sequence 2 is the sequence withthe 2^(nd) most abundant amino acid at a given position and so forth. Inone aspect of the invention, one or more (several) residues of SEQ IDNO: 3 are replaced by the corresponding residue from Consensus sequence1 or Consensus sequence 2 or Consensus sequence 3 or Consensus sequence4. In one aspect of the present invention the variants comprise analteration at one or more (several) of the positions selected from thegroup of 52 positions identified by the consensus sequence analysisconsisting of position number 10, 19, 68, 80, 89, 104, 111, 117, 123,129, 137, 139, 140, 147, 156, 159, 164, 165, 177, 179, 183, 200, 204,211, 222, 224, 225, 228, 232, 259, 267, 268, 269, 281, 328, 345, 366,374, 380, 383, 384, 406, 415, 436, 443, 445, 449, 450, 455, 456, 488 and507. In a preferred embodiment the alteration is a substitution, orseveral substitutions, selected from the group consisting of: I10V,D19E, Q68H, L80V, G89A, T104A, P111Q, A117S, S123P, K129T, Q137E, V139K,N140F, Q147S, R156Y, V159M, H164N, F165Y, A177T, V179I, A183S, G200P,G204T, R211K, I222V, A224P, G225S, V2281, V232A, V259I, R267K, L268K,S269A, F281 L, A328G, V3451, D366H, T374A, L380F, N383Y, D384G, K406N,N415G, H436Y, S443D, K445S, L449I, V4501, S455N, M456Y, K488T and P507A.

In another aspect of the invention the variant is generated by changingthose amino acids in the parental peptide which have a positive chargesand are situated within 20 Å of the calcium ion to neutral or negativecharged amino acids. Preferred variants of the present inventioncomprise variants in which the overall charge within 20 Å from thecalcium ion has been made more negative. In such variants positivelycharged amino acids may have been replaced with amino acids that areneutral or negatively charged under the application conditions. Inaccordance herewith, preferred variants may have an amino acid residuewhich is partly or fully positively charged under the “chemicalstability” or application conditions, i.e. a Lys, Arg or His replaced bya negative or neutral amino acid. Preferred replacement amino acids maybe negatively charged amino acids as Asp and Glu or neutral amino acidsas Ala, Asn, Gln, Tyr, Trp and Phe. A preferred variant of the presentinvention comprises an alteration at one or more of the positionsselected form the group consisting of position number 49, 87, 118, 129,134, 142, 156, 169 and 197. In a preferred embodiment the alterationsare substitutions at one or more of the positions selected form thegroup consisting of position number 87, 118, 129, 134, 142, 156, and169. In a preferred embodiment the substitution is selected from thegroup consisting of: K87A; K129A,S,F,I; K118A; K142A,Q,R156Y,F,V,I,K,W,L,M and K169Q,A.

In one aspect, a variant of a parent xyloglucanase comprises analteration at one or more (several) positions corresponding to positions68 or 123 or 156 or 118 or 200 or 129 or 137 or 193 or 92 or 76 or 331.Preferably, the variant comprises substitution at position 68 and one ormore substitutions at one or more additional positions, selected fromthe group consisting of position number 123, 156, 118, 200, 129, 137,193, 92, 83, 149, 34, 340, 332, 9, 76, 331, 310, 324, 498, 395 and 366.

In another aspect, a variant comprises a substitution at position 156and one or more substitutions at one or more additional positionsselected from the group consisting of position number 10, 13, 14, 19,37, 68, 78, 92, 118, 123, 129, 137, 139, 140, 147, 159, 164, 165, 169,176, 177, 179, 183, 200, 204, 211, 222, 224, 244, 247, 249, 259, 267,268, 269, 275, 288, 299, 301, 302, 303, 310, 324, 328, 331, 366, 380,383, 384, 389, 406, 409, 415, 436, 443, 445, 449, 450, 454, 455, 456,461, 470 and 507.

In another aspect, a variant of a parent xyloglucanase comprisesalterations at two or more (several) positions corresponding topositions 68 or 123 or 156 or 118 or 200 or 129 or 137 or 193 or 92 or76 or 331. Preferably, the variant comprises a substitution at position68 or 123 or 156 or 118 or 200 or 129. Even more preferably the variantcomprises a substitution at position 129 and position 156.

In another aspect, a variant of a parent xyloglucanase comprisesalterations at three or more (several) positions corresponding topositions 68 or 123 or 156 or 118 or 200 or 129 or 137 or 193 or 92 or76 or 331.

In another aspect, a variant of a parent xyloglucanase comprisesalterations at four or more (several) positions corresponding topositions 68 or 123 or 156 or 118 or 200 or 129 or 137 or 193 or 92 or76 or 331.

In another aspect, a variant of a parent xyloglucanase comprisesalterations at five or more (several) positions corresponding topositions 68 or 123 or 156 or 118 or 200 or 129 or 137 or 193 or 92 or76 or 331.

In another aspect, a variant of a parent xyloglucanase comprisesalterations at six or more (several) positions corresponding topositions 68 or 123 or 156 or 118 or 200 or 129 or 137 or 193 or 92 or76 or 331.

In another aspect, a variant of a parent xyloglucanase comprisesalterations at seven or more (several) positions corresponding topositions 68 or 123 or 156 or 118 or 200 or 129 or 137 or 193 or 92 or76 or 331.

In another aspect, a variant of a parent xyloglucanase comprisesalterations at the positions corresponding to positions 129 and 156 and331 and 200 and 118.

In another aspect, a variant of a parent xyloglucanase comprisesalterations at the positions corresponding to positions 68 and 129 and156 and 331 and 200 and 118.

In another aspect, a variant of a parent xyloglucanase comprisesalterations at the positions corresponding to positions 68 and 92 and129 and 156 and 331 and 200 and 118.

In another aspect the variant comprises one or more (several)substitutions selected from the group consisting of: Q68H,N,L; S123P,T;R156Y,F,V,I,K,W,L,M; KI 18A,R; G200P,E,S,D; K129T,A,S; Q137E; H193T,S,D;T92V,I,A,S; A83E; Q149E; L34F,I,V; R340T,N; S332P; T9D; S76W,V,I,K,R,T;N331F,C; M310I,V,L; D324N; G498A,D; D395G and D366H. Preferably, thesubstitutions are selected from the group consisting of Q68H; S123P;R156Y,F; K118A; G200P,E; K129T,A; Q137E; H193T; T92V and N331F. Morepreferably, the substitutions are selected from the group consisting ofQ68H; S123P; R156Y,F; K118A; G200P,E; K129T,A; Q137E; T92V and N331F.More preferably, the variant contains a substitution in nine or eight,seven or six or five or four or three or two or one position(s), wherethe substitutions are selected from the group consisting of Q68H; S123P;R156Y,F; K118A; G200P,E; K129T,A; Q137E; T92V and N331F.

In a further aspect the variant comprises one or more (several) of thefollowing combinations of substitutions:

Q68H; S123P; R156Y; Q68H+R156Y; K129A+R156Y; S123T+K129A+R156Y;K129A+R156Y+G200P; Q68H+K118R+R156F; Q68H+R156Y+H193T;Q68H+R156F+G200P+N331F; Q68H+T92V+K118A+R156Y;K118A+K129A+R156Y+G200P+N331F; G78A+T92V+K118A+K129A+R156Y;Q68H+K129T+R156K+G200P+N331F; K118A+K129A+R156Y+K169A+G200P+N331F;T92V+K118A+K129A+R156Y+G200P+N331F; G78A+K118A+K129A+R156Y+G200P+N331F;G78A+T92V+K118A+K129A+R156Y+K169A; Q68H+T92V+Q137E+R156Y+G200P+N331F;Q68H+T92V+K118A+Q137E+R156Y+N331F; Q68H+T92V+R156Y+G200P+M310V+N331F;Q68H+K118A+K129A+R156Y+G200P+N331F;Q68H+T92V+K118A+K129A+R156Y+G200P+N331F;Q68H+T92V+K118A+Q137E+R156Y+G200P+N331F;Q68H+T92V+K118A+K129A+R156Y+H193T+D366H;Q68H+T92V+K118A+K129A+Q137E+R156Y+H193T+D366H;Q68H+T92V+K118A+K129A+Q137E+R156Y+G200P+N331F;Q68H+T92V+K118A+S123P,T+K129A+Q137E+R156Y+G200P+N331F; orQ68H+T92V+K118A+K129A+Q137E+R156Y+G200P+A224P+N331F;

In a preferred embodiment all the variants described in the above arevariants of a parent xyloglucanase which belong to family 44 of glycosylhydrolases, more preferred the parent xyloglucanase is selected from axyloglucanase having at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95% identity to the amino acid sequence of SEQ ID NO: 3, morepreferred the parent xyloglucanase is selected from the group consistingof SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 and mostpreferred the parent xyloglucanases consists of SEQ ID NO: 3.

Polynucleotides

The present invention also relates to isolated polynucleotides thatencode variants of a parent xyloglucanase according to the presentinvention. In particular polynucleotides that encode a xyloglucanasevariant as described in the variant section above, is encompassed by thepresent invention. Polynucleotides of the invention will hybridize to adenatured double-stranded DNA probe comprising either the full variantsequence corresponding to positions 82-1653 of SEQ ID NO: 1 or position97 to 1653 of SEQ ID NO: 1 with proper sequence alterationscorresponding to actual amino acid alterations in the variant or anyprobe comprising a variant subsequence thereof having a length of atleast about 100 base pairs under at least medium stringency conditions,but preferably at high stringency conditions. The variantpolynucleotides of the present invention may also comprise silentmutations in addition to the mutations giving rise to the amino acidalterations described in the variant section above. Silent mutations aremutations in the three letter code which does not give rise to a changein the amino acid, e.g. GTT to GAT which both code for valine.

The polynucleotides encoding the xyloglucanase variants of the presentinvention include DNA and RNA. Methods for isolating DNA and RNA arewell known in the art. DNA and RNA encoding genes of interest can becloned in Gene Banks or DNA libraries by means of methods known in theart. Polynucleotides encoding polypeptides having xyloglucanase activityof the invention are then identified and isolated by, for example,hybridization or PCR.

Expression Vectors

The present invention also relates to expression vectors, in particularrecombinant expression vectors, comprising a nucleic acid construct ofthe invention. Nucleic acid constructs of the invention comprise anisolated polynucleotide encoding a variant xyloglucanase of the presentinvention, preferably operably linked to one or more control sequenceswhich direct the expression of the coding sequence in a suitable hostcell under conditions compatible with the control sequences. In creatingthe expression vector, the coding sequence is located in the vector sothat the coding sequence is operably linked with the appropriate controlsequences for expression. The control sequences may either be providedby the vector or by the nucleic acid construct inserted into the vector.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter may be any nucleotide sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell. Such promoters are well known in the art. The controlsequence may also be a suitable transcription terminator sequence, asequence recognized by a host cell to terminate transcription. Theterminator sequence is operably linked to the 3′ terminus of thenucleotide sequence encoding the polypeptide. Any terminator which isfunctional in the host cell of choice may be used in the presentinvention, such terminators are well known in the art. The controlsequence may also be a suitable leader sequence, a nontranslated regionof an mRNA which is important for translation by the host cell. Theleader sequence is operably linked to the 5′ terminus of the nucleotidesequence encoding the polypeptide. Any leader sequence that isfunctional in the host cell of choice may be used in the presentinvention, such leader sequences are well known in the art. The controlsequence may also be a signal peptide coding region that codes for anamino acid sequence linked to the amino terminus of a polypeptide anddirects the encoded polypeptide into the cell's secretory pathway. The5′ end of the coding sequence of the nucleotide sequence may inherentlycontain a signal peptide coding region naturally linked in translationreading frame with the segment of the coding region which encodes thesecreted polypeptide. Alternatively, the 5′ end of the coding sequencemay contain a signal peptide coding region which is foreign to thecoding sequence. The foreign signal peptide coding region may berequired where the coding sequence does not naturally contain a signalpeptide coding region. Alternatively, the foreign signal peptide codingregion may simply replace the natural signal peptide coding region inorder to enhance secretion of the polypeptide. However, any signalpeptide coding region which directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used in the presentinvention. The control sequence may also be a polyadenylation sequence,a sequence operably linked to the 3′ terminus of the nucleotide sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. It may also be desirable to add regulatorysequences which allow the regulation of the expression of thepolypeptide relative to the growth of the host cell. Examples ofregulatory systems are those which cause the expression of the gene tobe turned on or off in response to a chemical or physical stimulus,including the presence of a regulatory compound.

An isolated polynucleotide encoding a variant xyloglucanase of thepresent invention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotidesequence prior to insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotide sequences utilizing recombinant DNA methods are wellknown in the art. Furthermore, tags which may aid purification orimmobilization of the polypeptide may be added to the polypeptide. Sucha tag may for example be a polyhistidine tag (His tag). Preferably, thetag located in the N-terminal or C-terminal of the polypeptide, and maybe encoded by the vector. Alternatively, the tag may be locatedinternally in the polypeptide, as long as it does not affect thefunctionality of the polypeptide.

The recombinant expression vector may be any vector (e.g., a plasmid,phagemid, phage or virus) that can be conveniently subjected torecombinant DNA procedures and can bring about the expression of thenucleotide sequence. The choice of the vector will typically depend onthe compatibility of the vector with the host cell into which the vectoris to be introduced.

The vectors may be linear or closed circular plasmids. The vector may bean autonomously replicating vector, i.e., a vector that exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome.

The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers that permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers that conferantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof. Preferredfor use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention may contain an element(s) thatpermits stable integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

More than one copy of a nucleotide sequence of the present invention maybe inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleotide sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleotide sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleotide sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

In one embodiment of the present invention the plasmid vector maycontain the following elements:

i) a signal peptide coding region (e.g. obtained from the genes forBacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA), followed by a polynucleotidesequence encoding the mature xyloglucanase variant. This sequence may bepreceded by and operably linked to:

ii) a DNA sequence comprising a mRNA stabilising segment (e.g. derivedfrom the CryIIIa gene, as shown in WO 99/43835);

iii) a marker gene (e.g. a chloramphenicol resistance gene); and

iv) genomic DNA from Bacillus subtilis as 5′ and 3′ flanking segmentsupstream and downstream of the polynucleotide, respectively, to enablegenomic integration by homologous recombination between the flankingsegments and the Bacillus genome.

The vectors describe above may also be useful in the generation andscreening of the variants using the previously described mutagenesisprocedures.

Host Cells

The present invention also relates to recombinant a host cell comprisinga polynucleotide encoding a variant xyloglucanase of the invention,which are advantageously used in the recombinant production of thepolypeptides. A vector comprising a polynucleotide sequence of thepresent invention is introduced into a host cell so that the vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier.

The host cell may be a prokaryote such as bacterial cells, an archaea ora eukaryote such as fungal cells, plant cells, insect cells, ormammalian cells.

Useful prokaryotes are bacterial cells such as gram positive bacteriaincluding, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus halodurans,Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillusmegaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans orStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred embodiment, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred embodiment, the Bacilluscell is an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

In a preferred embodiment, the host cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK, page 171) and all mitosporic fungi (Hawksworth et al.,In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995,CAB International, University Press, Cambridge, UK). In a more preferredembodiment, the fungal host cell is a yeast cell. “Yeast as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport, eds,Soc. App. Bacteriol. Symposium Series No. 9, 1980).

In an even more preferred embodiment, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell. In a most preferred embodiment, the yeast host cell is aSaccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis or Saccharomyces oviformis cell. In another mostpreferred embodiment, the yeast host cell is a Kluyveromyces lactiscell. In another most preferred embodiment, the yeast host cell is aYarrowia lipolytica cell.

In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK). Thefilamentous fungi are characterized by a mycelial wall composed ofchitin, cellulose, glucan, chitosan, mannan, and other complexpolysaccharides. Vegetative growth is by hyphal elongation and carboncatabolism is obligately aerobic. In contrast, vegetative growth byyeasts such as Saccharomyces cerevisiae is by budding of a unicellularthallus and carbon catabolism may be fermentative. In an even morepreferred embodiment, the filamentous fungal host cell is a cell of aspecies of, but not limited to, Acremonium, Aspergillus, Fusarium,Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia,Tolypocladium, or Trichoderma. In a most preferred embodiment, thefilamentous fungal host cell is an Aspergillus awamori, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus nigeror Aspergillus oryzae cell. In another most preferred embodiment, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In an even most preferred embodiment, the filamentousfungal parent cell is a Fusarium venenatum (Nirenberg sp. nov.) cell. Inanother most preferred embodiment, the filamentous fungal host cell is aHumicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson and Simon, editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology 194: 182-187, Academic Press,Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; andHinnen et al., 1978, Proceedings of the National Academy of Sciences USA75: 1920.

A particular embodiment of the present invention is a recombinant hostcell transformed with a polynucleotide encoding a variant xyloglucanaseof the present invention. Preferably, such a host cell does not containan inherent xyloglucanase encoding gene, or such a gene has beendisrupted. Thereby the recombinant variant xyloglucanases is the onlyxyloglucanase produced by the recombinant host cell of the presentinvention.

Methods of Production

The present invention also relates to methods of producing axyloglucanase variant, comprising: (a) cultivating a host cell of thepresent invention under conditions suitable for the expression of thevariant; and (b) recovering the variant from the cultivation medium.

In the production methods of the present invention, the host cells arecultivated in a nutrient medium suitable for production of thexyloglucanase variant using methods known in the art. For example, thecell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

One embodiment of the present invention is a method of producing avariant of a parent xyloglucanase, wherein said variant hasxyloglucanase activity, said method comprising: a) culturing a cellunder conditions suitable for expression of the variant, where said cellcontains a polynucleotide sequence encoding a variant of a parentxyloglucanase in which said variant is altered in one or more (several)amino acid position(s) selected from the group consisting of positions:68, 123, 156, 118, 200, 129, 137, 193, 92, 83, 149, 34, 340, 332, 9, 76,331, 310, 324, 498, 395, 366, 1, 374, 7, 140, 8, 14, 21, 211, 37, 45,13, 78, 87, 436, 101, 104, 111, 306, 117, 119, 414, 139, 268, 142, 159,164, 102, 168, 176, 180, 482, 183, 202, 206, 217, 4, 222, 19, 224, 228,232, 2, 240, 244, 5, 247, 249, 328, 252, 259, 406, 267, 269, 275, 179,166, 278, 281, 288, 298, 301, 18, 302, 165, 80, 303, 316, 169, 322, 120,146, 342, 348, 147, 353, 380, 468, 382, 383, 38, 384, 389, 391, 10, 392,396, 177, 397, 399, 409, 237, 413, 253, 415, 418, 40, 443, 445, 148,449, 225, 450, 454, 3, 455, 456, 299, 461, 470, 204, 476, 488, 347, and507, and said polynucleotide sequence is prepared by mutagenesis of aparent polynucleotide sequence selected from the group consisting of SEQID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6, or a parent polynucleotidesequence having at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95% identity to the nucleotide sequence of SEQ ID NO: 1; and b)recovering the xyloglucanase variant from the cultivation medium.

In an alternative aspect, the xyloglucanase variant is not recovered,but rather a host cell of the present invention expressing a variant isused as a source of the variant.

The xyloglucanase variant may be detected using methods known in the artthat are specific for the expressed polypeptides. These detectionmethods may include use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the variant xyloglucanaseas described herein in the Examples.

The resulting xyloglucanase variant may be recovered by methods known inthe art. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,collection, centrifugation, filtration, extraction, spray-drying,evaporation, or precipitation.

A xyloglucanase variant of the present invention may be purified by avariety of procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure xyloglucanasevariants.

Compositions

The present invention also relates to compositions comprising a variantxyloglucanase or a polypeptide having xyloglucanase activity of thepresent invention. Preferably, the compositions are enriched in such avariant or polypeptide. The term “enriched” indicates that thexyloglucanase activity of the composition has been increased, e.g., withan enrichment factor of 1.1 or more. Preferably, the compositions areformulated to provide desirable characteristics such as low color, lowodor and acceptable storage stability.

The composition may comprise a variant or polypeptide of the presentinvention as the major enzymatic component, e.g., a mono-componentcomposition. Alternatively, the composition may comprise multipleenzymatic activities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a dryformulation. For instance, the polypeptide may be formulated in the formof a granulate or a microgranulate. The variant or polypeptide to beincluded in the composition may be stabilized in accordance with methodsknown in the art. In a preferred embodiment the variant xyloglucanase isformulated in a liquid composition.

Uses

The present invention is also directed to methods for using thexyloglucanase variants.

The variant xyloglucanases are preferably incorporated into and/or usedtogether with detergent compositions, for example in laundry detergentcompositions, for example household laundry detergent compositions,especially liquid laundry detergent compositions. The detergentcomposition typically comprises conventional detergent ingredients suchas surfactants (anionic, cationic, nonionic, zwitterionic, amphoteric),builders, bleaches, polymers, other enzymes and other ingredients, e.g.,as described in WO 2007/130562 and WO 2007/149806, which are herebyincorporated by reference in its entirety.

The detergent composition can be in any form, such as a solid, liquid,gel or any combination thereof, preferably the composition is in aliquid form, preferably a liquid laundry detergent composition.

An aspect of the invention is the use of a xyloglucanase variant or of axyloglucanase variant composition of the invention together with adetergent composition in order to impart de-pilling and/orfabric-softness and/or colour clarification and/or soil removal and/orsoil anti-redeposition and/or dye transfer inhibition benefits to afabric or garment.

Furthermore, the invention relates to a process for laundering offabrics comprising treating fabrics with a washing solution containing adetergent composition and a xyloglucanase variant or a xyloglucanasevariant composition of the invention. The laundering treatment can forexample be carried out in a machine washing process or in a manualwashing process. The washing solution can for example be an aqueouswashing solution containing the detergent composition and with a pHbetween 3 and 12.

During washing and use, the surface of fabrics or garment willconventionally become contaminated with broken or loosed fibre fragmentswhich can give the fabric a faded and worn appearance. Removal of thesesurface fibers from the fabric will partly restore the original coloursand looks of the fabric, resulting in colour clarification and enhancedappearance. A xyloglucanase variant or xyloglucanase variant compositionof the invention may be used to provide colour clarification and/orenhanced appearance by use in single or in multiple (repeated) washingcycles.

Furthermore, microfibrils protruding from the surface of the textile cangather into little balls, so-called pills or fluffs that stick to thesurface and disturb the appearance of the fabric. A xyloglucanasevariant or xyloglucanase variant composition of the invention may beused to remove such pills, an effect that is termed de-pilling.

Color-clarification and de-pilling can be assessed by visual inspectionusing a test group panel. The effects may also be measured by lightreflection or by determination of cotton fluffs by means of opticalmeasurements. These methods are generally known in the art and brieflydescribed in Enzymes in Detergency, 1997, published by Marcel Dekker,page 139 to page 140.

Especially with an increasing number of wash cycles, deposits, which caninclude particulate soils, soluble soils, dyes and pigments andinsoluble salts, build up on the textile fibre surfaces. This can leadsto a visible deterioration of the perceived cleaning performance of thewashing treatments for example leading to a greyish or yellowishappearance of the fabric. This may be prevented using a xyloglucanasevariant or xyloglucanase variant composition of the invention in thewash cycles. This effect is termed anti-redeposition or dye transferinhibition or soil removal and may be assessed by optical measurements.

Soil or insoluble salt particles trapped on the surface of the fabricand between the fibers can lead to stiffening of the fabric. Byincluding a xyloglucanase variant or xyloglucanase variant compositionof the invention in the wash cycles the fabric may be softened.

The fabrics subjected to the methods of the present invention may beconventional washable laundry, for example household laundry.Preferably, the major part of the laundry is garments and fabrics,including knits, wovens, denims, yarns, and towelling, made from cotton,cotton blends or natural or manmade cellulosics (e.g., originating fromwood pulp) or blends thereof. Examples of blends are blends of cotton orrayon/viscose with one or more companion material such as wool,synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyesterfibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyurethane fibers, polyurea fibers, aramid fibers), andcellulose-containing fibers (e.g. rayon/viscose, ramie, flax/linen,jute, cellulose acetate fibers, lyocell).

It is recognized that the treatment of fabrics and/or garments with adetergent solution containing the xyloglucanase variant or xyloglucanasevariant composition of the invention can be particularly relevant inconnection with, for example, production of new fibers and/or fabricsand/or garments, and also during laundering of used fabrics and/orgarments for example during household laundering processes or ininstitutional laundering processes.

The dosage of the xyloglucanase variant or the xyloglucanase variantcomposition of the present invention and other conditions, under whichthe composition is used, including the composition and concentration ofthe detergent solution, may be determined on the basis of methods knownin the art.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

The xyloglucanases can be used in the compositions of the presentinvention to effect removal of soils containing derivatives of celluloseor hemicellulose, enhance anti-redeposition and improve soil release.The xyloglucanses can also be used in the compositions of the presentinvention to impart soil release benefits to cotton during a subsequentlaundering process. The soil release benefit is observed on cottonfabric and on all types of fabric that comprise a significant amount ofcotton, such as cotton-synthetic (e.g. polyester, polyamide such asNylon™, and elastane) blends.

EXAMPLES Example 1 Production and Purification of Xyloglucanase Variants

The xyloglucanase variants of the present invention were prepared bystandard procedures, in brief: Introducing random and/or site-directedmutations into the gene, transforming Bacillus subtilis host cells withthe mutated genes, fermenting the transformed host cells, and obtainingthe xyloglucanase variant from the fermentation broth. The referencexyloglucanase (SEQ ID NO: 3) was produced recombinantly in Bacillussubtilis in a similar manner.

Fermentation was carried out in shake flask cultures at 37° C. for 4days shaking of 100 ml PS-1 medium containing one CaCO₃ tablet (0.5 g)in a baffled 500 ml Erlenmeyer flask. The PS-1 medium compositioncontains 100 g/L sucrose, 40 g/L Soymeal Meal, 10 g/L Na₂HPO₄*12H₂O, 0.1ml/L Dowfax 63N10 and antibiotic in the form of 6 μg/ml chloramphenicol.

After fermentation the culture broth was harvested by centrifugation(26000×g, 20 min). A small volume of the supernatant was sterilefiltered through a 0.45 μm filter, and stored frozen. The samples wereallowed to thaw immediately before the stability assays described belowwere started.

In some cases the enzyme samples were purified before they were used forthe stability test.

For enzyme purification the supernatants were filtered through a NALGENE0.2 μm Filtration unit (cat. no. 569-0020) in order to remove the restof the host cells. The pH of the 0.2 μm filtrate was adjusted to pH 5.0with 20% CH₃COOH and the filtrate was applied to an XpressLine ProAcolumn (UpFront chromatography A/S) equilibrated in 50 mM succinicacid/NaOH, 1 mM CaCl₂, pH 5.0. After washing the XpressLine ProA columnextensively with the equilibration buffer, the xyloglucanase was elutedby a step-elution with 50 mM Tris/HCl, pH 9.0. Fractions were collectedduring elution. Fractions from the column were analysed forxyloglucanase activity (Example 2) and fractions with activity werepooled. The pH of the pool was adjusted to pH 9.0 with 3 M Tris base andthe pool was diluted with demineralised water to the same (or lower)conductivity as 50 mM Tris/HCl, pH 9.0. The adjusted solution wasapplied to a SOURCE Q column (GE Healthcare) equilibrated in 50 mMTris/HCl, pH 9.0. After washing the SOURCE Q column extensively with theequilibration buffer, the enzyme was eluted with a linear NaCl gradient(0→0.5 M) in the same buffer over five column volumes. Fractions fromthe column were again analysed for xyloglucanase activity and activefractions were further analysed by SDS-PAGE. Fractions, where only oneband was seen on the Coomassie stained SDS-PAGE gel, were pooled as thepurified preparation.

Example 2 Xyloglucanase Assay

The xyloglucanase activity of enzyme samples, e.g. from purification,were measured in an AZCL-xyloglucan assay.

AZCL-xyloglucan (Megazyme) was incubated with the xyloglucanase and theliberated blue colour was measured at 650 nm. The xyloglucanase activitywas calculated as the increase in blue colour during incubation aftersubtraction of the proper blank value.

-   AZCL-xyloglucan substrate: 4 mg/ml AZCL-xyloglucan (Megazyme)    homogeneously suspended in 0.01% Triton X-100 by stirring.-   Assay temperature: 37° C.-   Assay buffer: 50 mM succinic acid/NaOH, 0.01% Triton X-100, pH 5.0.

500 μl AZCL-xyloglucan substrate suspension was placed on ice in anEppendorf tube. 500 μl Assay buffer was added and the mixture wasallowed to become ice-cold. 20 μl enzyme sample (diluted in 0.01% TritonX-100) was added. The assay was initiated by transferring the Eppendorftube to an Eppendorf thermomixer, which was set to the assaytemperature. The tube was incubated for 15 minutes on the Eppendorfthermomixer at its highest shaking rate (1400 rpm). The incubation wasstopped by transferring the tube back to the ice bath. When the tube hadbecome ice-cold, the tube was centrifuged shortly in an ice-coldcentrifuge to precipitate unreacted substrate. 200 μl supernatant wastransferred to a microtiter plate and A₆₅₀ was read. A buffer blank (20μl 0.01% Triton X-100 instead of enzyme) was included in the assay andthe difference in A₆₅₀ between enzyme sample and buffer blank was ameasure of the xyloglucanase activity.

Example 3 Stability of Xyloglucanase Variants

The detergent stability of the xyloglucanase variants of the presentinvention was assessed by measuring the activity of the variants afterincubation in a liquid detergent.

The stability test was performed by adding an enzyme sample into theliquid detergent and storing it at elevated temperatures, e.g., 35° C.or 40° C. After the prescribed storage time the enzyme activity wasdetermined and compared with the activity of an equivalent sample storedat approximately −18° C. for the same time period. The result of thestability test is the activity found in the sample stored at elevatedtemperature expressed as % of the activity found in the cold storedsample.

The results for the xyloglucanase variants were compared to the resultfor the parental xyloglucanase (SEQ ID NO:3), tested under the sameconditions. The ratio between these two stability results is theStability Improvement Factor (SIF).

Variants having a SIF>1 are more stable under the test conditions thanthe parental xyloglucanase. Preferred variants are those that have highSIF in this test.

Detergent

The liquid detergent used for the stability tests has the followingcomposition

alkylethoxy sulfate 20.1%  alkylbenzene sulfonate 2.7% alkyl sulfate6.5% alkyl ethoxylate 0.8% citric acid 3.8% fatty acid 2.0% Borax 3.0%Na & Ca formate 0.2% amine ethoxylate polymers 3.4%diethylenetriaminepentaacetic acid 0.4% Tinopal AMS-GX 0.2% Ethanol 2.6%Propylene glycol 4.6% Diethylene glycol 3.0% polyethylene glycol 0.2%Monoethanolamine 2.7% NaOH to pH 8.3 Minor ingredients (protease,amylase, 2.3% perfume, dye) Water balance

Storage Test

The enzyme samples prepared according to Example 1 were allowed to thawimmediately before starting the storage stability test.

The enzyme samples were diluted to a concentration of approximately 0.25mg enzyme protein per ml.

The liquid detergent was dispensed into glass bottles with a volume ofapproximately 12 ml, providing 1.0±0.05 gram of detergent in each glass.

For each enzyme sample two duplicate bottles were prepared. 50 μldiluted enzyme and a small magnetic stirrer bar was added to the bottlesand they were closed tightly (to prevent evaporation during storage).The contents were mixed with help of the magnetic stirrer bar for about5 minutes. One bottle of the pair was placed in a freezer atapproximately −18° C. The other bottle was placed in a suitableincubator oven at the prescribed elevated temperature, e.g. 35° C. or40° C., to be tested. After the prescribed storage time the bottles inthe incubator oven are transferred into the freezer.

Activity Assay

The activity of the enzyme samples after storage in detergent wasmeasured using the following procedure.

Materials and Reagents:

1 M phosphate buffer pH 7:Dissolve 138 grams of NaH₂PO₄.H₂O in about 750 ml water. Add 4 N NaOH togive pH 7.0. Then make the final volume to 1000 ml.Assay buffer (50 mM phosphate pH 7):Mix 950 ml water, 50 ml 1 M phosphate buffer pH7 and 5 ml of Berol 537(nonionic surfactant supplied by Akzo Nobel). Adjust the final pH to7.00±0.02.

Substrate:

Cellazyme C tablets, supplied by Megazyme International Ireland Ltd,catalogue number T-CCZ. The tablets contain cross-linked dyed HEcellulose.

Procedure

About 30 minutes prior to starting the assay the bottles weretransferred from the freezer into a refrigerator at approximately 4° C.Immediately before starting the assay the bottles were taken out of therefrigerator and placed on the laboratory bench top and opened.

10 ml assay buffer (room temperature) was added to each open bottle. Thebottles were then transferred into a 30° C. water bath equipped with asubmerged multipoint magnetic stirrer. The contents were stirred gentlyfor about 5 minutes.

One Cellazyme C tablet was added to each bottle. Stirring was continuedusing a stirrer speed which is just adequate to keep the substrateparticles in movement and avoid sedimentation. The bottles were removedfrom the water bath 30 minutes after addition of the tablet and werethen allowed to stand at room temperature with no stirring for 15minutes.

With a pipette approximately 1 ml of the practically clear supernatantfrom the top of each bottle was transferred into a semi-microspectrophotometer cuvette. Absorbance at 590 nm was then measured usinga suitable spectrophotometer. All measurements were finished within 15minutes.

Blank samples, i.e., equivalent detergent samples but containing noadded xyloglucanase enzyme, were included in the assay.

Calculation

For each enzyme sample there are two Abs590 measurements:

A590f, which is the Abs590 value of the sample stored at −18° C.

A590w, which is the Abs590 value of the sample stored at elevatedtemperature.

Subtract the blank value (A590b) from both A590f (giving A590f-A590b)and from A590w (giving A590w-A590b).

The stability was calculated as:

% Stability=((A590w−A590b)/(A590f−A590b))×100%.

For each enzyme the results for (A590f−A590b) must be in the range0.1-1.2. If the value is outside this range the result for that enzymemust be regarded as being unreliable and the test should be repeatedwith a different dilution of the enzyme sample.

Finally the Stability Improvement Factor (SIF) for each enzyme variantis calculated as follows: SIF=% stability of enzyme sample/% stabilityof parent enzyme (SEQ ID NO: 3)

Results

Below are the stability results of xyloglucanase variants tested underdifferent conditions.

TABLE 1 Sterile filtered enzyme samples stored for 18 hours at 40° C.Mutations SIF K8Q 1.1 K8A 1.2 K13A 1.1 K18R 1.1 K87Q 1.1 K129A 1.7 K169Q1.3 K169R 1.4 K169A 1.3 N140F 1.2 G316I 1.1 F418I 1.1 L34I 1.1 L166I 1.1L268I 1.1 L278I 1.3 V1* + V2* + H3* 1.2 *0aE + *0bV 1.3 F146L 1.2 Q137E1.6 R156Y 2.2 R156Q 1.5 K8S 1.2 K21T 1.4 K176P 1.1 K445S 1.4 K470T 1.2

TABLE 2 Purified enzyme samples stored for 18 hours at 40° C. MutationsSIF K87Q 1.1 K129A 1.8 K169A 1.1 A7T + G200P + A224P + G225K + R267K +L268K + S269A 1.3 H164N + V179I + G200A + R267K 1.2 H164N + V179I +G200A + R211K + G225D + F281L 1.5 H164N + G200A + G225N + R267K 1.2

TABLE 3 Sterile filtered enzyme samples stored for 24 hours at 40° C.Mutations SIF K101R + L102I 1.1 K217A 1.1 L380F 1.1 N383Y 1.2 G78A 1.2M310V 1.2 N399I 1.1 G498S 1.1 F146L 1.1 Q137E 1.4 R156Y 2.0 V1* + V2* +H3* + G4* + Q5* 1.1 N331F 1.2 K8S 1.1 T92V 1.3 K176P 1.2 G253A 1.1 K445S1.3 K470T 1.2

TABLE 4 Purified enzyme samples stored for 24 hours at 40° C. MutationsSIF T92V 1.2 Q137E 1.5 R156Y 1.7 R156Q 1.2

TABLE 5 Sterile filtered enzyme samples stored for 30 hours at 40° C.Mutations SIF K118R 1.1 K118A 1.7 K129A + K169A 1.6 G200P 1.5 K129A +R156Y 2.0 K129A + Q137E + R156Y 2.2 K129A + R156Y + H164N 2.1

TABLE 6 Purified enzyme samples stored for 30 hours at 40° C. MutationsSIF T92V 1.3 R156Y 1.9 K129A + R156Y 2.1

TABLE 7 Sterile filtered enzyme samples stored for 48 hours at 40° C.Mutations SIF K118A 3.0 K252Q 1.1 K252R 1.2 K252A 1.1 K275Q 1.1 K275R1.2 K275A 1.1 K306R 1.1 K306A 1.1 K347Q 1.1 K347R 1.1 K347A 1.1 K382A1.1 K414A 1.2 K445R 1.3 K454R 1.1 K476Q 1.1 K482Q 1.1 K482A 1.1 K488Q1.1 K488R 1.1 K488A 1.1 M40V 1.4 R156Y 2.9 G200P 1.8 K129A + R156Y 3.5K129A + Q137E + R156Y + K470T 3.7 K406N 1.1 K445S 1.2 K488T 1.2 T92V +K129A + R156Y 3.7 K118A + K129A + R156Y 3.8 T92V + K118A + K129A + R156Y3.9 K129A + R156Y + P507A 3.2 K129A + R156Y + S443D + K445S + L449I +V450I + S455N + 3.8 M456Y K129A + R156Y + H436Y 3.9 K129A + R156Y +K406N + N415G 3.5 K129A + R156Y + L380F + N383Y + D384G + N389T 3.5K129A + R156Y + D366H + T374A 3.4 K129A + R156Y + A328G 3.5 K129A +R156Y + V259I + R267K + L268K + S269A 3.5 K129A + R156Y + T244D 3.4K129A + R156Y + I222V + A224P + V228I + V232A 2.0 K129A + R156Y +G200P + G204T + R211K 3.6 K129A + R156Y + A177T + V179I + A183S 2.9K129A + R156Y + V159M + H164N + F165Y 2.8 K129A + R156Y + I10V + V14I +D19E 4.0 T104A + P111Q + A117S + K129A + R156Y 2.1 S123T + K129A + R156Y3.8 K129A + Q137E + V139K + N140F + Q147S + R156Y 2.9 K129A + R156Y +D324N 3.4 K129A + R156Y + K176P 3.2 K129A + R156Y + D249N 3.2 K129A +R156Y + D249G 3.3 K129A + R156Y + D249S 3.1 K129A + R156Y + D461N 3.6K129A + R156Y + D461T 3.9 K129A + R156Y + D461Q 4.0 K129A + R156Y +R409T 3.8 K129A + R156Y + R409L 3.6 K129A + R156Y + D247G 1.4 K129A +R156Y + E288Q 2.7 D37G + K129A + R156Y 3.9 D37N + K129A + R156Y 3.6K129A + R156Y + R267H 3.8 K129A + R156Y + D303I 4.1 K129A + R156Y +D303K 3.7 K129A + R156Y + K275T 3.5 K129A + R156Y + G200P 3.9 K129A +R156Y + N331F 3.8 R156Y + N331F 3.2 K118A + K129A + R156Y + K470T 4.4K470R 1.1 K470P 1.2 G413A 1.1 K118A + K129A + R156Y + A224P 3.9 D119L1.3 K87V + K129A + K169A 1.9 K129A + K445S 1.8 K118A + K129A + R156Y +G200P 3.8 K118A + K129A + R156Y + G200P + N331F 4.2 G78A + K118A +K129A + R156Y 3.8 G78A + T92V + K118A + K129A + R156Y 3.8 T92V + K118A +K129A + R156Y 3.7 M310V + N399I 1.7 L34I + K129A 1.9 K101A + K129A 1.8K13A + K129A 2.0 K129A + K470T 1.8 K129A + K176P 1.9 G78A + T92V +K118A + K129A + R156Y + K169A 4.8 K118A + K129A + R156Y + K169A +G200P + N331F 4.7 K118A + K129A + R156Y + G200P + M310V + N331F 4.7K129A + R156Y + K454Q 3.8 G78A + K118A + K129A + R156Y + G200P + N331F4.2 T92V + K118A + K129A + R156Y + G200P + N331F 4.3 K129A + R156Y +N302K + D303S 2.9 K129A + R156Y + N302K + D303L 2.7 S332P + V397I 1.1K129A + R156Y + K322I + K454Q 2.3 Q68H + K118A + K129A + R156Y + G200P +N331F 4.1 Q68H + T92S + K118A + K129A + R156Y + G200P + N331F 5.2 Q68H +T92A + K118A + K129A + R156Y + G200P + N331F 4.7 Q68H + K118A + K129A +R156Y + G200P + N331F 5.0 Q68H + K118A + K129A + R156Y + G200P + N331F5.7 Q68H + T92D + K118A + K129A + R156Y + G200P + N331F 3.3 Q68H +T92I + K118A + K129A + R156Y + G200P + N331F 4.4 Q68H + K118A + K129A +R156Y + G200P + N331F 4.4 Q68H + T92V + K118A + K129A + R156Y + G200P +N331F 4.2 K129S 1.1 K129A 1.5 R156M 1.3 R156F 2.3 R156W 1.6 R156L 1.4R156V 2.2 G396P 1.3 G413S 1.1 A177T 1.1 E38I 1.1 E38V 1.2 G36V + D37A +E38* + N39* 1.2 T104A 1.2 L102A + T104V + *104P 1.3 Q68L 1.3 Q68H 3.6N389A 1.1 G468Y 1.1 G237V 1.1

TABLE 8 Purified enzyme samples stored for 48 hours at 40° C. MutationsSIF K118A 2.3 R156Y 2.5 K129A + K169A 1.7 G200P 1.5 K129A + R156Y 1.7K129A + Q137E + R156Y 3.7 K129A + R156Y + H164N 3.5 K129A + Q137E +R156Y + K470T 4.2 T92V + K129A + R156Y 4.5 K118A + K129A + R156Y 3.8K129A + R156Y + G200P 4.8 K129A + R156Y + N331F 4.1 R156Y + N331F 3.5K118A + K129A + R156Y + G200P, 4.2 K118A + K129A + R156Y + G200P + N331F4.5 G78A + K118A, + K129A + R156Y 4.0 G78A + T92V + K118A + K129A +R156Y 4.3 Q68H 3.7

TABLE 9 Sterile filtered enzyme samples stored for 72 hours at 40° C.Mutations SIF K13R 1.3 K206Q 1.1 K129A + R156Y 5.1 K129A + Q137E +R156Y + K470T 6.4 T92V + K129A + R156Y 6.6 K118A + K129A + R156Y 7.2K129A + R156Y + G200P 7.7 K129A + R156Y + N331F 5.9 R156Y + N331F 5.3

TABLE 10 Sterile filtered enzyme samples stored for one week at 35° C.Mutations SIF K8Q 1.4 K8A 1.1 K13Q 1.1 K18Q 1.1 K18A 1.4 K21Q 1.4 K21R1.4 K21A 1.4 K87Q 1.3 K101R 1.3 K101A 1.6 K118R 1.4 K118A 2.3 K101R +L102I 1.1 K129A 2.1 K169Q 1.4 K169R 1.5 K169A 1.5 K220Q 1.3 K220A 1.2K252Q 1.1 K252R 1.1 K275Q 1.1 K275R 1.1 K275A 1.1 K306R 1.1 K306A 1.1K307Q 1.2 K307R 1.1 K454Q 1.6 K454R 1.2 K476Q 1.3 K476R 1.3 K476A 1.2K482Q 1.2 K482A 1.2 K488Q 1.2 K488R 1.2 K488A 1.1 N140F 1.7 G78A 1.2M310V 1.3 G316I 1.1 W391V 1.1 N399I 1.4 L34I 1.3 L268I 1.1 L278I 1.2G498S 1.2 *0aE + *0bV 1.4 F146L 2.3 Q137E 2.0 R156Y 3.2 R156Q 1.7 N331F1.5 K8S 1.3 K21T 1.5 K176P 1.2 G253A 1.1 K445S 1.5 K470T 1.6 F146C 1.3K129A + K169A 1.8 G200P 1.7 A224P 1.1 K129A + R156Y 2.6 K129A + Q137E +R156Y 2.6 K129A + R156Y + H164N 2.6 K406N 1.3 K445S 1.2 K488T 1.2 K129R1.1 R156F 2.0

TABLE 11 Purified enzyme samples stored for one week at 35° C. MutationsSIF K101R 1.1 K101A 1.1 K118A 2.3 K129A 1.8 K169R 1.2 K169A 1.1 T92V 2.0F418I 1.1 V1* + V2* + H3* + G4* + Q5*; 1.2 Q137E 1.6 R156Y 2.5 R156Q 1.2K21T 1.1 G200P 1.7 K129A + R156Y 2.7 K129A + Q137E + R156Y 3.0 K129A +R156Y + H164N 3.1 A7T + G200P + A224P + G225K + R267K + L268K + S269A1.3 H164N + V179I + G200A + R267K 1.3 H164N + V179I + G200A + R211K +G225D + F281L 1.8 H164N + G200A + G225N + R267K 1.6

TABLE 12 Purified enzyme samples stored for 16 hours at 44° C. MutationSIF Q68H 5.8 S123P 4.4 R156Y 4.0 K118A 2.9 G200P 2.6 K129A 2.4 Q137E 2.4H193T 2.1 T92V 2.0 S76W 1.7

Example 4 Stability of Xyloglucanase Variants

The detergent stability of the xyloglucanase variants of the presentexample was assessed by measuring the activity of the variants afterincubation in a liquid detergent.

The stability test was performed by adding an enzyme sample into theliquid detergent and storing it at elevated temperatures, e.g. 35° C. or46° C. After the prescribed storage time the enzyme activity wasdetermined and compared with the activity of an identical sample thathad been stored cold at approximately +5° C. for the same time period.The result of the stability test is the activity found in the samplestored at elevated temperature (the stressed sample) expressed as % ofthe activity found in the equivalent cold-stored sample (the unstressedsample).

The results for the xyloglucanase variants were compared to the resultfor the parental xyloglucanase (SEQ ID NO:3), tested under the sameconditions.

Detergent

The liquid detergent used for the stability tests has the followingcomposition

alkylethoxy sulfate 20.1%  alkylbenzene sulfonate 2.7% alkyl sulfate6.5% alkyl ethoxylate 0.8% citric acid 3.8% fatty acid 2.0% Borax 3.0%Na & Ca formate 0.2% amine ethoxylate polymers 3.4%diethylenetriaminepentaacetic acid 0.4% Tinopal AMS-GX 0.2% Ethanol 2.6%Propylene glycol 4.6% Diethylene glycol 3.0% polyethylene glycol 0.2%Monoethanolamine 2.7% NaOH to pH 8.3 Minor ingredients (protease,amylase, 2.3% perfume, dye) Water balance

Storage Test

The enzyme samples prepared according to Example 1 were allowed to thawimmediately before starting the storage stability test.

The enzyme samples were used without further dilution.

The liquid detergent was dispensed into a round-bottom polystyrene96-well microtiter plate (Plate 1) providing 190 μl of detergent perwell.

Ten μl enzyme sample and a small magnetic stirrer bar was added to eachwell and the plate was closed tightly (to prevent evaporation) usingadhesive aluminium foil lids (Beckman Coulter). The contents were mixedwith the magnetic stirrer bars for about 30 minutes.

From each well of Plate 1, 20 μl detergent-enzyme mixture was thentransferred into a new empty identical plate (Plate 2). Both plates werethen sealed.

The original plate (Plate 1) was placed in an incubator oven at theprescribed elevated temperature, e.g. 35° C. or 46° C., to be tested.The other plate (Plate 2) was placed in a refrigerator at approximately5° C.

Following incubation for the prescribed period, the plates were removedfrom the refrigerator and the incubator oven. The plates were placed onthe laboratory bench for at least half an hour to allow all wells toreach room-temperature.

Then 20 μl from each well of Plate 1 was transferred into a new emptyround bottom 96-well plate (Plate 1a).

Plate 1a now contains 20 μl stressed samples and Plate 2 contains 20 μlunstressed samples.

Activity Assay

The activity of the enzyme samples after storage in detergent wasmeasured using the following procedure at room temperature.

Assay Principle:

Para-nitrophenol-beta-D-cellotetraoside (pNP-beta-D-cellotetraoside) isa synthetic substrate that is hydrolysed by the catalytic action ofcertain xyloglucanase enzymes.

The substrate itself is colourless; however upon hydrolysis of theterminal reducing end glycoside bond, para-nitrophenol is released whichis yellow in a pH8 buffer due to a strong absorbance at 405 nm.

pNP-beta-D-cellotetraoside itself is very stable under the given assayconditions. Thus increasing absorbance at 405 nm is an attribute ofenzymatic activity.

We found that the parental xyloglucanase (SEQ ID NO:3) acceptedpNP-beta-D-cellotetraoside as substrate, as evidenced by the strongabsorbance increase at 405 nm.

Materials and Reagents: Assay Buffer: 100 mM EPPS; 0.01% Tween 20; pH8.0.

pNP-beta-D-cellotetraoside (CAS-#: 129411-62-7; Toronto ResearchChemicals; Canada)Substrate solution: 1 mM pNP-beta-D-cellotetraoside in assay buffer.

Procedure:

Plate 1a contains 20 μl stressed samples and Plate 2 contains 20 μlunstressed samples.

The samples were diluted by adding 50 μl assay buffer to all wells inPlate 1a and Plate 2, and mixed for one hour using a microtiter plateshaker. Then an additional 50 μl assay buffer was added to all wells andthe shaking was continued for an additional 10 minutes.

20 μl of the factor 6 diluted samples were transferred to a transparent384 well polystyrene microtiter plate, and 20 μl substrate solution wasadded to all wells. The samples were mixed by shaking the microtiterplate briefly. The kinetic measurement of enzymatic activity wasinitiated immediately by observing the rate of increasing absorbance at405 nm using a 384-well spectrophotometric reader.

The initial velocity (Abs/min) of the reaction was determined. Theinitial velocity of the reaction was a measure of the enzymatic activityin the sample as verified by a linear standard curve within relevantenzyme concentrations.

Calculation:

% residual activity was calculated as enzymatic activity in the stressedsample divided by enzymatic activity in the identical unstressed sample.

% residual activity=“Abs/min(stressed sample)”/“Abs/min(not stressedsample)”*100%.

Results

Below are the stability results of xyloglucanase variants tested underdifferent conditions.

TABLE 13 Sterile filtered enzyme samples stored for 16 hours at +44° C.% Residual Mutations Activity SEQ ID NO: 3 7 K118A 24 R156Y 36 K129A +K169A 19 G200P 26 K129A + R156Y 51 K129A + Q137E + R156Y 72 K129A +R156Y + H164N 63

TABLE 14 Sterile filtered enzyme samples stored for 16 hours at +47° C.% Residual Mutations Activity SEQ ID NO: 3 <5 Q68H + T92S + K118A +K129A + R156Y + G200P + 77 N331F Q68H + T92A + K118A + K129A + R156Y +G200P + 83 N331F Q68H + K118A + K129A + R156Y + G200P + N331F 91 Q68H +T92D + K118A + K129A + R156Y + G200P + 49 N331F Q68H + T92Y + K118A +K129A + R156Y + G200P + 78 N331F Q68H + T92I + K118A + K129A + R156Y +G200P + 89 N331F Q68H + T92V + K118A + K129A + R156Y + G200P + 95 N331FQ68H + T92S + K118A + K129A + R156Y + G200P + 67 G274D + N331F Q68H +T92N + D97N + K118A + K129A + R156Y + 81 G200P + N331F Q68H 52 K118A +K129A + R156Y 52 T92V + K118A + K129A + R156Y 88 K129A + R156Y + G200P +G204T + R211K 68 S123T + K129A + R156Y 65 K129A + R156Y + G200P 73K118A + K129A + R156Y + G200P + N331F 90 G78A + K118A + K129A + R156Y +G200P + N331F 98 T92V + K118A + K129A + R156Y + G200P + N331F 95

TABLE 15 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 22 R156Y 59 K13R 34 K307Q 31K414A 34 G253A 33 G498S 31 M310V 38 N399I 30 V1* + V2* + H3* + G4* + Q5*31 F146L 34 K445S 30 K470T 30

TABLE 16 Sterile filtered enzyme samples stored for 16 hours at +45° C.% Residual Mutations Activity SEQ ID NO: 3 6 R156Y 34 K129A + R156Y 55K101R + L102I 12 K118A + K129A + R156Y 72 K129A + R156Y + P507A 57K129A + R156Y + D366H + T374A 44 K129A + R156Y + V259I + R267K + L268K +S269A 40 K129A + R156Y + G200P + G204T + R211K 49 K129A + R156Y +V159M + H164N + F165Y 30 T104A + P111Q + A117S + K129A + R156Y 39S123T + K129A + R156Y 70 K129A + R156Y + D324N 60 K129A + R156Y + D461N59 K129A + R156Y + D461T 61 K129A + R156Y + D461Q 59 D37G + K129A +R156Y 60 D37N + K129A + R156Y 64 K129A + R156Y + R267H 64 K129A +R156Y + D303I 62 K129A + R156Y + D303K 65 K129A + R156Y + K275T 68K129A + R156Y + G200P 92 K118A + K129A + R156Y + K470T 80 H164N <5K129A + R156Y + N302K + D303S 66 K129A + R156Y + N302K + D303L 64

TABLE 17 Sterile filtered enzyme samples stored for 16 hours at +44° C.% Residual Mutations Activity SEQ ID NO: 3 26 R156Y 58 K118A + R156Y +G200P 84 K118A + K129A + Q137E + R156Y + G200P + N331F 92 K445C + K470C32 F281L 32 D366H 35 K392G 26 D395G 35 S76W 47 G498D 32 G498A 36 D324N39 S123T 36 Q68Y 6 Q68C 13 K129A + R156Y 89 K118A + K129A + R156Y +G200P + N331F 100

TABLE 18 Sterile filtered enzyme samples stored for 16 hours at +44° C.% Residual Mutations Activity SEQ ID NO: 3 34 R156Y 66 R156M 39 R156F 63R156W 44 R156L 34 R156P <5 R156V 50 R156T 35 R156S 27 R156A 36 R156D 34R156K 52 R156N 29 R156I 50 T92I 39 R156Q 34

TABLE 19 Sterile filtered enzyme samples stored for 16 hours at +44° C.% Residual Mutations Activity SEQ ID NO: 3 25 R156Y 70 R156E 66 R156F 65T92V 43 R156P <5 R156V 53 R156K 38 R156I 31

TABLE 20 Sterile filtered enzyme samples stored for 16 hours at +44° C.% Residual Mutations Activity SEQ ID NO: 3 31 R156Y 65 N415S 34 S443E 33S443K 32 S443Q 35 K129T 46 K129A 50 G468Y 32 G237A 34 G237S 34 G237V 25G468S 32

TABLE 21 Sterile filtered enzyme samples stored for 16 hours at +44° C.% Residual Mutations Activity SEQ ID NO: 3 21 R156Y 45 S332P 41 K129A +R156Y + K176S 73 K129A + R156Y + D303V 77 K129A + R156Y + D303S 81 R197L20 R340N 41 R340T 43 H193S 51 H193D 49 H193T 66 L34F 43 Q137D 24 Q149E48 T9D 40 A83E 49 S214E 25 K129A + R156Y 98 T92V 49 T92I 36

TABLE 22 Sterile filtered enzyme samples stored for 16 hours at +47° C.% Residual Mutations Activity SEQ ID NO: 3 <5 R156Y 29 Q68H + R156V +G200P + N331F 93 Q68H + R156F + G200P + N331F Approx. 100 Q68H + G200P +N331F Approx. 100 Q68H + T92V + R156V + G200P + M310V 86 Q68H + T92V +R156Y + G200P + M310V 86 Q68H + T92V + R156F + G200P + M310V 91 Q68H +T92V + R156F + G200P + M310V + S484C 82 Q68H + T92V + G200P + M310V 82Q68H + T92V + R156V + G200P + M310V + N331F Approx. 100 Q68H + T92V +R156Y + G200P + M310V + N331F Approx. 100 Q68H + T92V + R156F + G200P +M310V + N331F 86 Q68H + T92V + G200P + M310V + N331F 80 D366H <5 K118A +K129A + R156Y + G200P + N331F 81 Q68H + K118A + K129A + R156Y + G200P +N331F 87 Q68H + T92V + K118A + K129A + R156Y + G200P + 80 N331F M40L +A41T + Q67M + N72S + S76D + G78A + 41 Q82K + Q137E + N153K + H164N +D249N + V272A + I337L + M356L + V397A + N415S + T421I + S424N + N441D +V450I + E489A + A490V + T517A + S522* I10V + F17S + D33E + M40L + Q67M +N72S + S76D + 52 G78A + Q82K + T92A + L102Q + Q137E + I222V + V228I +D249N + V272A + I337L + M356L + T374A + V397A + S416A + T421I + S424N +N441D + D444Y + V450I + A469E + K470T + I473G + T517A + S522P + P523V +V524E Q32H + M40L + R49G + D65E + Q67M + N72S + 41 S76D + G78A + Q82K +92A + L102Q + T104A + Q137E + H164N + K202E + I222V + V228I + D249N +M356L + T374A I10V + F17S + Y53H + Q67M + N72S + S76D + G78A + 26 Q82K +T92A + L102Q + Q137E + T172V + A177T + I222V + V228I + D249N + S269N +I337L + M356L + V397A + S416A + T421I + S424H + N441D + D444Y + A469E +K470T + I473G + T517A + S522*

TABLE 23 Sterile filtered enzyme samples stored for 64 hours at +46° C.% Residual Mutations Activity SEQ ID NO: 3 <5 R156Y <5 Q68H + R156V +G200P + N331F 80 Q68H + R156F + G200P + N331F 84 Q68H + G200P + N331F 63Q68H + T92V + R156V + G200P + M310V 52 Q68H + T92V + R156Y + G200P +M310V 67 Q68H + T92V + R156F + G200P + M310V 63 Q68H + T92V + R156F +G200P + M310V + S484C 68 Q68H + T92V + G200P + M310V 48 Q68H + T92V +R156V + G200P + M310V + N331F 93 Q68H + T92V + R156Y + G200P + M310V +N331F 100 Q68H + T92V + R156F + G200P + M310V + N331F 91 Q68H + T92V +G200P + M310V + N331F 80 K118A + K129A + R156Y + G200P + N331F 56 Q68H +K118A + K129A + R156Y + G200P + N331F 86 Q68H + T92V + K118A + K129A +R156Y + G200P + 88 N331F

TABLE 24 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 16 R156Y 52 T374A 27 F146L +K322I 24 K129A + Q137E + R156Y + G200P 87 Q68S 14 Q68T <5 K129A + R156Y71 F146L 26 K129A + R156Y + G200P 82 Q68H 77

TABLE 25 Sterile filtered enzyme samples stored for 16 hours at +44° C.% Residual Mutations Activity SEQ ID NO: 3 19 R156Y 53 K101A + K129A 47K129A + K470T 46 S332P 29 G413A 30 K118A + K129A + R156Y + A224P 81K129A + K176P 50 K118A + K129A + R156Y + K169A + G200P + N331F 89K118A + K129A + R156Y + G200P + M310V + N331F 86 K129A + R156Y + K454Q86 K13A + K129A 49 G78A + T92V + K118A + K129A + R156Y + K169A 93K129A + R156Y + K322I + K454Q 76 K129A 47 K129A + R156Y 74 K118A +K129A + R156Y 77 K118A + K129A + R156Y + G200P + N331F Approx. 100G78A + T92V + K118A + K129A + R156Y 93

TABLE 26 Sterile filtered enzyme samples stored for 6 days at +46° C. %Residual Mutations Activity SEQ ID NO: 3 <5 R156Y <5 Q68H + R156V +G200P + N331F 50 Q68H + R156Y + G200P + N331F 60 Q68H + R156F + G200P +N331F 64 Q68H + G200P + N331F 40 Q68H + T92V + R156V + G200P + M310V 32Q68H + T92V + R156Y + G200P + M310V 42 Q68H + T92V + R156F + G200P +M310V 43 Q68H + T92V + R156F + G200P + M310V + S484C 34 Q68H + T92V +G200P + M310V 27 Q68H + T92V + R156F + G200P + M310V + N331F 93 Q68H +T92V + G200P + M310V + N331F 58 K118A + K129A + R156Y + G200P + N331F 27Q68H + K118A + K129A + R156Y + G200P + N331F 75 Q68H + T92V + K118A +K129A + R156Y + G200P + 70 N331F

TABLE 27 Sterile filtered enzyme samples stored for 64 hours at +44° C.% Residual Mutations Activity SEQ ID NO: 3 <5 R156Y 9 K101A + K129A 6K129A + K470T 4 S332P <5 G413A <5 K118A + K129A + R156Y + A224P 51K129A + K176P 6 K118A + K129A + R156Y + K169A + G200P + N331F 67 K118A +K129A + R156Y + G200P + M310V + N331F 63 K129A + R156Y + K454Q 52 K13A +K129A 5 G78A + T92V + K118A + K129A + R156Y + K169A 72 K129A 5 K129A +R156Y 32 K118A + K129A + R156Y 30 K118A + K129A + R156Y + G200P + N331F63 G78A + T92V + K118A + K129A + R156Y 72

TABLE 28 Sterile filtered enzyme samples stored for 64 hours at +46° C.Mutations % Residual Activity SEQ ID NO: 3 <5 R156Y 4 G78A + T92V +K118A + K129A + R156Y + G200P + N331F 71 K118A + K129A + R156Y + G200P +N331F + N399I 59 K118A + K129A + F146L + R156Y + G200P + N331F 62 T92V +K118A + K129A + Q137E + R156Y + G200P + N331F 74 T92V + K118A + K129A +R156Y + H164N + G200P + N331F 70 Q68H + T92V + K118A + K129A + Q137E +R156Y + G200P + N331F 87 Q68H + T92V + K118A + S123T + K129A + Q137E +R156Y + G200P + N331F 90 T92V + K118A + K129A + R156Y + G200P + N331F 66K118A + K129A + R156Y + G200P + N331F 68 Q68H T92V K118A K129A R156YG200P N331F 83

TABLE 29 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 19 R156Y 51 S123P 69 V159M 21V345I 34 G225S 30 V232A <10

TABLE 30 Sterile filtered enzyme samples stored for 10 days at +46° C.Mutations % Residual Activity SEQ ID NO: 3 <5 R156Y <5 G78A + T92V +K118A + K129A + R156Y + G200P + N331F 32 K118A + K129A + R156Y + G200P +N331F + N399I 16 K118A + K129A + F146L + R156Y + G200P + N331F 23 T92V +K118A + K129A + Q137E + R156Y + G200P + N331F 34 T92V + K118A + K129A +R156Y + H164N + G200P + N331F 31 Q68H + T92V + K118A + K129A + Q137E +R156Y + G200P + N331F 67 Q68H + T92V + K118A + S123T + K129A + Q137E +R156Y + G200P + N331F 81 T92V + K118A + K129A + R156Y + G200P + N331F 23K118A + K129A + R156Y + G200P + N331F 25 Q68H T92V K118A K129A R156YG200P N331F 61

TABLE 31 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 15 R156Y 51 Q68F <5 Q68N 69Q68Y <5 Q68D <10 Q68C <10 Q68G <10 Q68S <10 Q68E <5 Q68A <5 Q68M 27 Q68W<10 Q68H 82

TABLE 32 Sterile filtered enzyme samples stored for 7 days at +46° C. %Residual Mutations Activity SEQ ID NO: 3 <5 R156Y <5 Q68H + T92V +K118A + K129A + 81 Q137E + R156Y + G200P + A224P + N331F Q68H + T92V +K118A + Q137E + 74 R156Y + G200P + N331F Q68H + T92V + Q137E + R156Y +G200P + N331F 80 Q68H + T92V + K118A + Q137E + G200P + N331F 65 Q68H +T92V + K118A + Q137E + R156Y + N331F 80 Q68H + T92V + K118A + Q137E +R156Y + G200P 67 G78A + K118A + K129A + R156Y + K169A 14 Q68H + T92V +K118A + K129A + Q137E + 73 R156Y + G200P + N331F K129A + R156Y <5 G78A +K118A + K129A + R156Y 7

TABLE 33 Sterile filtered enzyme samples stored for 48 hours at +46° C.Mutations % Residual Activity SEQ ID NO: 3 <5 R156Y 9 K118A + K129A +R156Y + G200P + N331F 67 Q68H + K118A + K129A + R156Y + G200P + N331F 79Q68H + T92V + K118A + K129A + R156Y + G200P + N331F 85 Q68H + T92V +K118A + K129A + R156Y + H193T + D366H 73 Q68H + T92V + K118A + K129A +Q137E + R156Y + H193T + D366H 72 Q68H + T92V + R156Y + H193T + D366H 78Q68H + T92V + R156F + H193T + D366H 78 Q68H + R156Y + H193T + D366H 68Q68H + T92V + K118A + K129A + R156Y + H193T 67 Q68H + T92V + K118A +K129A + Q137E + R156Y + H193T 80 Q68H + T92V + R156Y + H193T 84 Q68H +T92V + R156F + H193T 66 Q68H + R156Y + H193T 66 Q68H + R156Y + H193T +G200P + M310V 93 Q68H + T92V + R156F + H193T + G200P + M310V 82 Q68H +T92V + K118A + K129A + Q137E + R156Y + H193T + G200P + 76 M310V + E446KQ68H + T92V + R156Y + H193T + G200P + M310V 73 Q68H + T92V + K118A +K129A + R156Y + H193T + G200P + M310V 89 Q68H + K129T + R156K + G200P +N331F 95 Q68H + K129A + R156K + G200P + N331F 86 Q68H + K118A + R156V +G200P + N331F 81 Q68H + K118S + R156F + G200P + G274D + N331F 68

TABLE 34 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 22 R156Y 61 S123T + K129A +R156Y 83 H193T 44 G78A + T92V + K118A + 91 K129A + R156Y S123T 55 S123P73 V232A <10 K129A + R156Y 64 K118A + K129A + R156Y 68

TABLE 35 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 17 R156Y 60 N140F 25 H164A 7H193A 23 R500T 30 R500A 33 R500V 29 H199A <10 H3A 26 H436A 26 H448A <10H512A 25 H96A 14 H3A + H436A 27

TABLE 36 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 27 R156Y 66 N399I 33 L34F 35Q149E 35 S332P 36 K129A 50 K21Q + K129A 54 K129A + K275Q 56 Q68F 6 T9D +L34F + A83E + Q149E + 53 H193T + S332P + R340T

TABLE 37 Sterile filtered enzyme samples stored for 12 days at +37° C.Mutations % Residual Activity SEQ ID NO: 3 <5 R156Y 8 K118A + K129A +R156Y + G200P + N331F 52 Q68H + K118A + K129A + R156Y + G200P + N331F 47Q68H + T92V + K118A + K129A + R156Y + G200P + N331F 67 Q68H + R156Y +G200P + N331F 47 Q68H + R156F + G200P + N331F 66 Q68H + T92V + R156Y +G200P + M310V 41 Q68H + T92V + K118A + K129A + R156Y + H193T + D366H 54Q68H + T92V + K118A + K129A + Q137E + R156Y + H193T + D366H 44 Q68H +T92V + R156Y + H193T + D366H 44 Q68H + T92V + R156F + H193T + D366H 37Q68H + R156Y + H193T + D366H 36 Q68H + T92V + K118A + K129A + R156Y +H193T 50 Q68H + T92V + K118A + K129A + Q137E + R156Y + H193T 56 Q68H +T92V + R156Y + H193T 37 Q68H + T92V + R156F + H193T 37 Q68H + R156Y +H193T 44 Q68H + R156Y + H193T + G200P + M310V 34 Q68H + T92V + R156F +H193T + G200P + M310V 28 Q68H + T92V + K118A + K129A + Q137E + R156Y +H193T + G200P + 47 M310V + E446K Q68H + T92V + R156Y + H193T + G200P +M310V 47 Q68H + T92V + K118A + K129A + R156Y + H193T + G200P + M310V 56

TABLE 38 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 19 R156Y 49 G200S 28 G200D 25G200Y 12 G200L <5 G200P 37 G200W <5 G200I <5 G200N 9 G200F <5 G200V 9G200H 12 G200Q 19 G200C 17 G200A 24 G200M 6 G200K 11 G200E 48 G200R <5G200T 5

TABLE 39 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 13 R156Y 45 K21Q + K129A 34K129A + K275Q 39 T9D + L34F + A83E + Q149E + 43 H193T + S332P + R340TN399I 24 L34F 22 Q149E 23 S332P 24 K129A 58 G518D 19 K118A + K129A 73K118A 48 K129A + K169A 40

TABLE 40 Purified enzyme samples stored for 5 days at +46° C. Mutations% Residual Activity SEQ ID NO: 3 <5 R156Y <5 Q68H + T92V + K118A +K129A + 73 Q137E + R156Y + H193T + D366H Q68H + R156Y + H193T 63 Q68H 13Q68H + T92V + K118A + 70 Q137E + R156Y + N331F G78A + T92V + K118A +K129A + R156Y 44 K118A + K129A + R156Y + G200P + 46 N331F Q68H + T92V +K118A + K129A + 83 R156Y + G200P + N331F Q68H + K129T + R156K + G200P +N331F 77 Q68H + T92V + K118A + K129A + 85 R156Y + H193T + D366H

TABLE 41 Sterile filtered enzyme samples stored for 5 days at +46° C.Mutations % Residual Activity SEQ ID NO: 3 <5 R156Y <5 Q68H + T92V +K118A + K129A + Q137E + R156Y + H193T + N331K 70 Q68H + T92V + K118A +K129A + Q137E + R156Y + H193T + N331H 42 Q68H + T92V + K118A + K129A +Q137E + R156Y + H193T + N331Q 24 Q68H + T92V + K118A + K129A + Q137E +R156Y + H193T 33 Q68H + K118A + Q137E + R156Y + G200P + N331F 74 Q68H +S76W + T92V + K118A + Q137E + R156Y + G200P + N331F 87 K13A + Q68H +T92V + K118A + Q137E + R156Y + G200P 54 Q68H + T92V + K118A + Q137E +R156Y + G200P + D324N 53 Q68H + T92V + K118A + Q137E + R156Y + G200P +K470T 69 Q68H + T92V + K118A + Q137E + R156Y + G200P + N331F 75 Q68H +T92V + K118A + Q137E + R156Y + G200P 52

TABLE 42 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 13 R156Y 43 S76M 21 S76I 36S76E 19 S76R 26 S76K 27 S76V 39 S76R 24

TABLE 43 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 20 R156Y 51 K118A + R156Y 62R197A <5 R20A 26 R267A 26 R295A 23 R314A <10 R340A 23 A221K 25 M290R 23M373Q 25 V397S 25 T417K 27 N441G + A442E + S443D 30 S467R + G468S +A469T 29 I473T 24 A490R 32 T517A + G518D 31 V431E 29 S76W + G200P +A224P 58 S76W + G200P 59 G200P + A224P 56 S76T 42 M310V 31 G200P 47G200E 59 M310V + N399I <10 Q68W <5

TABLE 44 Sterile filtered enzyme samples stored for 16 hours at +46° C.Mutations % Residual Activity SEQ ID NO: 3 8 R156Y 40 Q68H + T92V +K118A + Q137E + 89 N140F + R156Y + G200P + K470T Q68H + T92V + K118A +S123P + 88 K129A + Q137E + R156Y + G200P + N331F T92V + K118A + Q137E +88 R156Y + G200P + N331F S76W + G200P + A224P 44 S76W + G200P 45 G200P +A224P 48 S76T 26 Q68H + T92V + K118A + Q137E + 91 R156Y + G200P + M310LQ68H + T92V + K118A + K129A + 95 Q137E + R156Y + G200P + N331F G200P 39

TABLE 45 Sterile filtered enzyme samples stored for 9 days at +46° C.Mutations % Residual Activity SEQ ID NO: 3 <5 R156Y <5 Q68H + T92V +K118A + K129A + Q137E + R156Y + H193T + N331K 46 Q68H + T92V + K118A +K129A + Q137E + R156Y + H193T + N331H 19 Q68H + T92V + K118A + K129A +Q137E + R156Y + H193T + N331Q 9 Q68H + T92V + K118A + K129A + Q137E +R156Y + H193T 17 Q68H + K118A + Q137E + R156Y + G200P + N331F 48 Q68H +S76W + T92V + K118A + Q137E + R156Y + G200P + N331F 65 K13A + Q68H +T92V + K118A + Q137E + R156Y + G200P 31 Q68H + T92V + K118A + Q137E +R156Y + G200P + D324N 30 Q68H + T92V + K118A + Q137E + R156Y + G200P +K470T 41 Q68H + T92V + K118A + Q137E + R156Y + G200P + N331F 50 Q68H +T92V + K118A + Q137E + R156Y + G200P 30

TABLE 46 Purified enzyme samples stored for 9 days at +46° C. Mutations% Residual Activity SEQ ID NO: 3 <5 R156Y <5 Q68H + T92V + K118A +K129A + 52 Q137E + R156Y + H193T + D366H Q68H + R156Y + H193T 34 Q68H +T92V + K118A + 45 Q137E + R156Y + N331F G78A + T92V + K118A + K129A +R156Y 14 K118A + K129A + R156Y + G200P + 18 N331F Q68H + T92V + K118A +K129A + 56 R156Y + G200P + N331F Q68H + K129T + R156K + G200P + N331F 47Q68H + T92V + K118A + K129A + 52 R156Y + H193T + D366H Q68H + R156Y +H193T 31

TABLE 47 Sterile filtered enzyme samples stored for 30 days at +37° C.Mutations % Residual Activity SEQ ID NO: 3 <5 R156Y <5 K118A + K129A +R156Y + G200P + N331F 33 Q68H + K118A + K129A + R156Y + G200P + N331F 42Q68H + T92V + K118A + K129A + R156Y + G200P + N331F 52 Q68H + R156Y +G200P + N331F 41 Q68H + R156F + G200P + N331F 58 Q68H + T92V + R156Y +G200P + M310V 41 Q68H + T92V + R156F + G200P + M310V 42 Q68H + T92V +K118A + K129A + R156Y + H193T + D366H 50 Q68H + T92V + K118A + K129A +Q137E + R156Y + H193T + D366H 32 Q68H + T92V + R156Y + H193T + D366H 33Q68H + T92V + R156F + H193T + D366H 28 Q68H + R156Y + H193T + D366H 25Q68H + T92V + K118A + K129A + R156Y + H193T 41 Q68H + T92V + K118A +K129A + Q137E + R156Y + H193T 43 Q68H + T92V + R156Y + H193T 27 Q68H +T92V + R156F + H193T 23 Q68H + R156Y + H193T 33 Q68H + R156Y + H193T +G200P + M310V 28 Q68H + T92V + R156F + H193T + G200P + M310V 21 Q68H +T92V + K118A + K129A + Q137E + R156Y + H193T + G200P + 310V + 35 E446KQ68H + T92V + R156Y + H193T + G200P + M310V 35 Q68H + T92V + K118A +K129A + R156Y + H193T + G200P + M310V 46

TABLE 48 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 15 R156Y 49 A83S 15 A83N 9A83Y 10 A83H 14 A83I 8 A83L 10 A83R 16 A83D 17 A83T 12 A83E 31 L34V 22L34M 19 L34I 24 M310I 21 M310V 20 M310L 18

TABLE 49 Sterile filtered enzyme samples stored for 3 days at +35° C.Mutations % Residual Activity SEQ ID NO: 3 61 R156Y 89 N331K 57 N331R 54N331L 39 N331H 62 N331G 59 N331M 70 N331W 55 N331S 58 N331V 57 N331T 46N331Y 55 N331I 47 N331A 87 N331Q 82 N331C 70 N331E 58 N331D 63 N331P 26N331F 51

TABLE 50 Sterile filtered enzyme samples stored for 16 hours at +44° C.Mutations % Residual Activity SEQ ID NO: 3 20 R156Y 58 I10V + F17S +Q67M + N72S + S76D + G78A + Q82K + T104A + Q137E + 72 N153K + R156Q +V219A + I222V + V228I + D249N + S269N + V272A + E333A + I337L + M356L +V397A + N415S + D420G + T421I + S424H + N441D + D444Y + V450I + A469E +K470T + I473G + T517A + S522* I10V + D33E + M40L + A41T + Q67M + Y73F +S76D + G78A + Q82K + 71 T92A + L102Q + Q137E + I222V + V228I + D249N +S269N + V272A + E333A + I337L + M356L + T374A + S416A + D444Y + A469E +K470T + I473G + T517A + S522* I10V + F17S + D33E + M40L + Q67M + N72S +S76D + G78A + Q82K + 78 T92A + L102Q + Q137E + H164N + N168K + T172A +V219A + I222V + V228I + D249N + S269N + V272A + E333A + I337L + M356L +N415S + T421I + S424H + N441D + D444Y + S522P + P523V + V524E I10V +F17S + D33E + Q67M + N72S + S76D + G78A + Q82K + T92A + 74 L102Q +Q137E + N168K + T172A + I222V + V228I + D249N + V272A + E333A + I337L +M356L + V397A + S416A + T421I + S424H + N441D + D444Y + A469E + K470T +I473S + V477I + E489A + A490V + T517A + S522* I10V + F17S + M40L +Q67M + N72S + S76D + G78A + Q82K + T92A + 73 L102Q + Q137E + I222V +V228I + D249N + S269N + V272A + T320A + I337L + M356L + T374A + V397A +N415S + T421I + S424H + N441D + D444Y + A469E + K470T + I473S + V477I +T517A + S522P + P523V + V524E I10V + F17S + D33E + M40L + A41T + Q67M +N72S + S76D + G78A + 64 Q82K + Q137E + V219A + D249N + V272A + I337L +M356L + V397A + S416A + T421I + S424N + N441D + D444Y + V450I + K470T +I473S + V477I I10V + F17S + Q67M + N72S + S76D + G78A + Q82K + T92A +T104A + 66 Q137E + R156Q + V159A + H164N + N168K + T172A + I222V +V228I + D249N + V272A K118A + K129A + R156Y + G200P + N331F 98 Q68H +T92V + K118A + K129A + R156Y + G200P + N331F Approx 100

TABLE 51 Sterile filtered enzyme samples stored for 2 days at +44° C.Mutations % Residual Activity SEQ ID NO: 3 <5 R156Y 20 Q68H + R156Y 61Q68H + T92V + K118A + R156Y 66 Q68H + T92V + R156Y 68 Q68H + K118A +R156Y + H193T + 74 D366H Q68H + T92V + K118R + 65 R156Y + H193T + D366HQ68H + T92V + K118R + R156F 63 Q68H + K118R + R156Y 68 Q68H + T92V +R156Y + H193T + D366H 69 Q68H + K118R + R156Y + G200P 74 Q68H + K118R +R156F 66 K118A + K129A + R156Y + G200P + 79 N331F Q68H + T92V + K118A +91 K129A + R156Y + G200P + N331F Q68H 55 D33V + Q68H + N168H + V450I 70S123T 10 K129A 10

1. An isolated variant of a parent xyloglucanase, the variant comprisingan alteration of the parent xyloglucanase at one or more positionsselected from the group consisting of position number 68, 123, 156, 118,200, 129, 137, 193, 92, 83, 149, 34, 340, 332, 9, 76, 331, 310, 324,498, 395, 366, 1, 374, 7, 140, 8, 14, 21, 211, 37, 45, 13, 78, 87, 436,101, 104, 111, 306, 117, 119, 414, 139, 268, 142, 159, 164, 102, 168,176, 180, 482, 183, 202, 206, 217, 4, 222, 19, 224, 228, 232, 2, 240,244, 5, 247, 249, 328, 252, 259, 406, 267, 269, 275, 179, 166, 278, 281,288, 298, 301, 18, 302, 165, 80, 303, 316, 169, 322, 120, 146, 342, 348,147, 353, 380, 468, 382, 383, 38, 384, 389, 391, 10, 392, 396, 177, 397,399, 409, 237, 413, 253, 415, 418, 40, 443, 445, 148, 449, 225, 450,454, 3, 455, 456, 299, 461, 470, 204, 476, 488, 347, and 507, whichposition corresponds to a position in amino acid sequence SEQ ID NO:3and wherein a) the alteration(s) are i) an insertion of an amino aciddownstream of the amino acid which occupies the position, and/or ii)deletion of the amino acid which occupies the position, and/or iii) asubstitution of the amino acid which occupies the position with adifferent amino acid; b) the parent xyloglucanase is a family 44xyloglucanase; and c) the variant has xyloglucanase activity. 2-23.(canceled)